Adeno-Associated-Virus Rep Sequences, Vectors and Viruses

ABSTRACT

The invention provides adeno-associated virus (AAV) replication (Rep) sequences. In one embodiment, the invention provides nucleotide sequences encoding a chimeric protein, wherein the encoded chimeric protein contains a wild type AAV Rep inhibitory amino acid sequence, and wherein the nucleotide sequences contain a scrambled and/or deoptimized polynucleotide sequence encoding the wild type AAV Rep inhibitory amino acid sequence. The invention provides vectors, cells, and viruses containing the invention&#39;s sequences. Also provided are methods for detecting portions of the AAV Rep inhibitory amino acid sequence, which reduce replication and/or infection and/or productive infection by viruses. The invention&#39;s compositions and methods are useful for site-specific integration and/or expression of heterologous sequences by recombinant adeno-associated virus (rAAV) vectors and by rAAV virus particles, such as hybrid viruses (e.g., Ad-AAV) comprising such vectors. The invention&#39;s compositions and methods find application in, for example, gene therapy and/or vaccines.

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 16/254,758, filed on Jan. 23, 2019, whichissued as U.S. Pat. No. 10,947,514 on Mar. 16, 2021, which is acontinuation of U.S. patent application Ser. No. 14/795,214, filed onJul. 9, 2015, which issued as U.S. Pat. No. 10,214,730 on Feb. 26, 2019,which is a continuation of U.S. patent application Ser. No. 14/112,703,filed on Nov. 19, 2013, now abandoned, which is the U.S. National Entryof International Application No. PCT/US12/34247, filed on Apr. 19, 2012,now abandoned, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 61/476,858, filed on Apr. 19, 2011, nowabandoned, each of which is herein incorporated by reference in itsentirety.

This invention was made with government support under grant AI041636,awarded by the National Institutes of Health (NIH). The government hascertain rights in the invention.

A Sequence Listing has been submitted in an ASCII text file named“17324ST25” created on Jun. 25, 2021, consisting of 35,028 bytes, theentire content of which is herein incorporated by reference.

FIELD OF INVENTION

The invention provides adeno-associated virus (AAV) replication (Rep)sequences. In one embodiment, the invention provides nucleotidesequences encoding a chimeric protein, wherein the encoded chimericprotein contains a wild type AAV Rep inhibitory amino acid sequence, andwherein the nucleotide sequences contain a scrambled and/or deoptimizedpolynucleotide sequence encoding the wild type AAV Rep inhibitory aminoacid sequence. The invention provides vectors, cells, and virusescontaining the invention's sequences. Also provided are methods fordetecting portions of the AAV Rep inhibitory amino acid sequence, whichreduce replication and/or infection and/or productive infection byviruses. The invention's compositions and methods are useful forsite-specific integration and/or expression of heterologous sequences byrecombinant adeno-associated virus (rAAV) vectors and by rAAV virusparticles, such as hybrid viruses (e.g., Ad-AAV) comprising suchvectors. The invention's compositions and methods find application in,for example, gene therapy and/or vaccines.

BACKGROUND

Sustained phenotypic correction of genetic defects requires a safe meansof gene replacement. To date, many of these gene correction strategiesuse integrating lentiviruses or retroviruses for long-term genereplacement, although their clinical applications remain limited becauseof potential for viral-associated oncogenesis.

Gene correction strategies have attempted to use hybridAdenovirus/Adeno-associated viruses (Ad/AAV) to combine the capacity,tropism and ease of production of adenovirus (Ad) with adeno-associatedvirus's (AAV's) ability for site-specific integration (SSI) intochromosome 19 AAVS1. Although the AAV Rep78 protein is required for SSI,the AAV Rep78 protein has the disadvantage of an inhibitory effect on Adreplication, particularly when co-expressed within the Ad backbone. Thishas lead to difficulty in the prior art in generating an integratingtransgene within the back-bone of a single hybrid virus, such as Ad/AAV.

While an Adenovirus carrying the AAV cis acting elements can beconstructed, construction of an Adenovirus carrying the Rep expressioncassette has met with only limited success. Work by various authors hasshown that coinfection with AAV in general, and Rep protein expressionin particular, results in a 10% to 40% decrease in Adenoviralreplication. Further, during co-infection of Ad and AAV, Rep protein hasbeen shown to co-localize to Adenoviral replication centers and preventtheir maturation. As a result, strategies to construct an Ad/AAVcarrying Rep have focused on controlling Rep expression. The fewsuccesses reported have utilized tightly regulated expression systems,within a helper dependent Adenoviral vector. Although these vectors arefree of adenoviral genes, they however need a helper virus forreplication. Also, construction of a first generation Adenoviruscarrying Rep has proved to be more difficult. Several reports exist ofunsuccessful strategies for the construction of a first generation Adcarrying Rep.

In particular, a stable first generation adenovirus carrying AAV Rep78has so far not been reproducibly constructed. Most viruses either failto grow, showing no signs of viral replication (Ueno et al. (2000)Biochemical and Biophysical Research Communications 273(2):473-478),grow slowly, or are unstable, acquiring deletions within the Rep gene(Zolotukhin (2005) Human Gene Therapy 16(5):551-557). In one report,analysis of two clones bearing deletions revealed no overlap of thedeletion sites within the Rep ORF (Zhang et al. (2001) Gene Ther.8:704).

Thus there remains a need for new compositions and methods for safe,site-specific gene integration for applications that include genetherapy, vaccine, etc.

SUMMARY OF THE INVENTION

The invention provides a recombinant nucleotide sequence encoding achimeric protein, a) wherein the encoded chimeric protein i) comprisesat least a portion of wild type AAV Rep inhibitory amino acid sequencelisted as SEQ ID NO:20, and ii) has Rep-mediated nuclease activity, andb) wherein the recombinant nucleotide sequence comprises a scrambledpolynucleotide sequence encoding the at least portion of the wild typeAAV Rep inhibitory amino acid sequence listed as SEQ ID NO:20.

In one embodiment, the nucleotide sequence further comprises aheterologous polynucleotide sequence operably linked to a first AAV ITR.In a preferred embodiment, the heterologous polynucleotide sequence isflanked by the first AAV ITR and by a second AAV ITR. In an alternativeembodiment, the heterologous polynucleotide sequence comprises atherapeutic sequence, exemplified by a therapeutic sequence that encodesone or both of a disease associated polypeptide and an antigenpolypeptide. In one embodiment, the nucleotide sequence furthercomprises a nucleic acid sequence encoding an AAV capsid protein. Inanother embodiment, the scrambled polynucleotide sequence comprises atleast a portion of SEQ ID NO:18. In a more preferred embodiment, theportion of SEQ ID NO:18 comprises SEQ ID NO:07. In an alternativeembodiment, the scrambled polynucleotide sequence comprises adeoptimized AAV Rep inhibitory nucleotide sequence. In a particularlypreferred embodiment, the deoptimized AAV Rep inhibitory nucleotidesequence comprises at least a portion of SEQ ID NO:19. In yet anotherembodiment, the portion of SEQ ID NO:19 comprises SEQ ID NO:09. In someembodiments, the scrambled polynucleotide sequence is operably linked toa promoter.

The invention also provides, an expression vector comprising any one ormore of the recombinant nucleotide sequences described herein.

Also provided by the invention is a recombinant adeno-associated virus(rAAV) comprising any one or more of the recombinant nucleotidesequences described herein. In a particular embodiment, the rAAV isinfectious, and more preferably (though not necessarily) the infectiousrAAV is replication competent. Yet more preferably (though notnecessarily) the replication competent rAAV is productive. In oneembodiment, the rAAV is produced by a permissive cell at substantiallythe same copy number as the copy number of a control AAV that lacksexpression of AAV Rep protein. In some embodiments, the rAAV ischaracterized by site-specific integration into adeno-associated virusintegration site 1 (AAVS1) sequence. In alternative embodiments, therAAV expresses at least a functional portion of Rep78 protein SEQ IDNO:04 at a reduced level compared to the level expressed by a controlhybrid virus that comprises wild type amino acid sequence SEQ ID NO:20that is encoded by the wild type AAV Rep inhibitory nucleotide sequencelisted as SEQ ID NO:17. In a more preferred embodiment, the rAAV is ahybrid virus that comprises at least a portion of a heterologous virusgenome sequence. In some embodiments, the heterologous virus is selectedfrom the group of adenovirus, herpes simplex virus, retrovirus,lentivirus, and baculovirus.

The invention also provides a cell comprising any one or more of therecombinant nucleotide sequences described herein.

Also provided by the invention is a composition comprising any one ormore of the recombinant adeno-associated virus (rAAV) described herein,wherein the composition is free of helper virus. In a preferredembodiment, the composition is a vaccine that comprises at least onepharmaceutically acceptable compound selected from the group of diluent,carrier, excipient, and adjuvant.

The invention additionally provides a method for detecting a sequencethat reduces replication by a virus, comprising a) providing i) a firstexpression vector comprising a first nucleotide sequence comprising ascrambled polynucleotide sequence encoding a portion of wild type AAVRep inhibitory amino acid sequence listed as SEQ ID NO:20, ii) a secondexpression vector comprising a second nucleotide sequence, wherein thesecond nucleotide sequence is produced by substituting a portion of thescrambled polynucleotide sequence with a corresponding portion of wildtype AAV Rep inhibitory nucleotide sequence listed as SEQ ID NO:17, andiii) a host cell that is permissive for the virus, b) transfecting i)the first expression vector into the permissive cell under conditions toproduce a first virus that comprises a first amino acid sequence encodedby the first nucleotide sequence, and ii) the second expression vectorinto the permissive cell under conditions to produce a second virus thatcomprises a second amino acid sequence encoded by the second nucleotidesequence, and c) determining the level of replication of the first virusand of the second virus in the transfected permissive cell, wherein areduced level of replication of the second virus compared to the firstvirus identifies the portion of wild type AAV Rep inhibitory nucleotidesequence as reducing replication by the virus. In one embodiment, theportion of SEQ ID NO:17 comprises SEQ ID NO:01. In another embodiment,the portion of SEQ ID NO:17 comprises one or more of SEQ ID NO:01, SEQID NO:21, SEQ ID NO:24 and SEQ ID NO:27.

The invention also provides a method for detecting a sequence thatreduces replication by a virus, comprising a) providing i) a firstexpression vector comprising a first nucleotide sequence comprising aportion of wild type AAV Rep inhibitory nucleotide sequence listed asSEQ ID NO:17, ii) a second expression vector comprising a secondnucleotide sequence, wherein the second nucleotide sequence is producedby substituting the portion of the wild type AAV Rep inhibitorynucleotide sequence with a scrambled polynucleotide sequence encodingthe portion of the wild type AAV Rep inhibitory nucleotide sequence, andiii) a host cell that is permissive for the virus, b) transfecting i)the first expression vector into the permissive cell under conditions toproduce a first virus that comprises a first amino acid sequence encodedby the first nucleotide sequence, and ii) the second expression vectorinto the permissive cell under conditions to produce a second virus thatcomprises a second amino acid sequence encoded by the second nucleotidesequence, and c) determining the level of replication of the first virusand of the second virus in the transfected permissive cell, wherein anincreased level of replication of the second virus compared to the firstvirus identifies the portion of the wild type AAV Rep inhibitorynucleotide sequence as reducing replication by the virus. In oneembodiment, the portion of SEQ ID NO:17 comprises SEQ ID NO:01. In afurther embodiment, the portion of SEQ ID NO:17 comprises one or more ofSEQ ID NO:01, SEQ ID NO:21, SEQ ID NO:24 and SEQ ID NO:27.

Further provided by the invention is a method for producing arecombinant adeno-associated virus (rAAV) particle, comprising a)providing an expression vector comprising any one or more of therecombinant nucleotide sequences described herein, b) providing anadeno-associated virus (AAV) packaging cell, and c) transfecting thepackaging cell with the expression vector to produce a recombinantadeno-associated virus (rAAV). In one embodiment, the method furthercomprises detecting the presence of the produced recombinantadeno-associated virus (rAAV). In another embodiment, the method furthercomprises isolating the produced recombinant adeno-associated virus(rAAV). In yet a further embodiment, the method does not includetransfecting the packaging cell with a helper virus.

The invention also provides a recombinant adeno-associated virus (rAAV)produced by any one or more of the methods described herein.

The invention additionally provides a method for reducing one or moresymptoms of disease in a mammalian subject, comprising administering atherapeutically effective amount of any one or more of the vectorsdescribed herein to a mammalian subject in need of the therapy. In oneembodiment, the method further comprises detecting the presence of atleast a portion of the vector in a cell of the treated subject. In analternative embodiment, the recombinant nucleotide sequence furthercomprises a heterologous polynucleotide sequence operably linked to afirst adeno-associated virus inverted terminal repeat (AAV ITR). In aparticular embodiment, the heterologous polynucleotide sequencecomprises a therapeutic sequence, exemplified by a therapeutic sequencethat encodes one or both of a disease associated polypeptide and anantigen polypeptide. In some embodiments, the therapeutic sequenceencodes an antigen polypeptide, and the method further comprisesdetecting an immune response by the subject to the antigen polypeptide.

The invention also provides a recombinant nucleotide sequence encoding achimeric protein, a) wherein the encoded chimeric protein i) compriseswild type AAV Rep inhibitory amino acid sequence ii) has Rep-mediatednuclease activity, and b) wherein the recombinant nucleotide sequencecomprises a scrambled polynucleotide sequence encoding the wild type AAVRep inhibitory amino acid sequence. In one embodiment, the wild type AAVRep inhibitory amino acid sequence comprises and/or consists of SEQ IDNO:22. In another embodiment, the wild type AAV Rep inhibitory aminoacid sequence comprises and/or consists of SEQ ID NO:25. In a furtherembodiment, the wild type AAV Rep inhibitory amino acid sequencecomprises and/or consists of SEQ ID NO:28. In one embodiment, thescrambled polynucleotide sequence encoding the wild type SEQ ID NO:22comprises SEQ ID NO:23. In another embodiment, the scrambledpolynucleotide sequence encoding the wild type SEQ ID NO:25 comprisesSEQ ID NO:26. In a further embodiment, the scrambled polynucleotidesequence encoding the wild type SEQ ID NO:28 comprises SEQ ID NO:29. Ina particular embodiment, the scrambled polynucleotide sequence comprisesa deoptimized AAV Rep inhibitory nucleotide sequence.

The invention additionally provides an expression vector comprising anyone or more of the recombinant nucleotide sequences disclosed herein.

The invention also provides a recombinant adeno-associated virus (rAAV)comprising any one or more of the recombinant nucleotide sequencesdisclosed herein. In one embodiment, the rAAV is infectious. In afurther embodiment, the infectious rAAV is replication competent. In aparticular embodiment, the replication competent rAAV is productive. Inyet another embodiment, the rAAV is produced by a permissive cell atsubstantially the same copy number as the copy number of a control AAVthat lacks expression of AAV Rep protein. In a further embodiment, therAAV is characterized by site-specific integration into adeno-associatedvirus integration site 1 (AAVS1) sequence. In another embodiment, therAAV expresses Rep78 protein SEQ ID NO:04 at a reduced level compared tothe level expressed by a control hybrid virus that comprises wild typeamino acid sequence SEQ ID NO:20 that is encoded by the wild type AAVRep inhibitory nucleotide sequence listed as SEQ ID NO:17. In anotherembodiment, the rAAV is a hybrid virus that comprises at least a portionof a heterologous virus genome sequence, exemplified by genomes ofadenovirus, herpes simplex virus, retrovirus, lentivirus, and/orbaculovirus.

The invention also provides a cell comprising any one or more of therecombinant nucleotide sequences described herein.

Also provided by the invention is a composition comprising any one ormore of the recombinant adeno-associated virus (rAAV) described herein,wherein the composition is free of helper virus. In a particularembodiment, the composition is a vaccine that comprises at least onepharmaceutically acceptable compound selected from the group consistingof diluent, carrier, excipient, and adjuvant.

The invention additionally provides a method for producing a recombinantadeno-associated virus (rAAV) particle, comprising a) providing anexpression vector comprising any one or more of the recombinantnucleotide sequences described herein, b) providing an adeno-associatedvirus (AAV) packaging cell, and c) transfecting the packaging cell withthe expression vector to produce a recombinant adeno-associated virus(rAAV). In a particular embodiment, the method further comprisesdetecting the presence of the produced recombinant adeno-associatedvirus (rAAV). In a further embodiment, the method further comprisesisolating the produced recombinant adeno-associated virus (rAAV). In analternative embodiment, the method does not include transfecting thepackaging cell with a helper virus. The invention also provides arecombinant adeno-associated virus (rAAV) produced by the invention'smethod.

Also provided herein is a method for reducing one or more symptoms ofdisease in a mammalian subject, comprising administering atherapeutically effective amount of any one or more of the vectorsdisclosed herein to a mammalian subject in need of the therapy. In oneembodiment, the method further comprises detecting the presence of atleast a portion of the vector in a cell of the treated subject. In afurther embodiment, the recombinant nucleotide sequence furthercomprises a heterologous polynucleotide sequence operably linked to afirst adeno-associated virus inverted terminal repeat (AAV ITR). In yetanother embodiment, the heterologous polynucleotide sequence comprises atherapeutic sequence, exemplified by a therapeutic sequence that encodesone or both of a disease associated polypeptide and an antigenpolypeptide. In a further embodiment, the therapeutic sequence encodesan antigen polypeptide, and the method further comprises detecting animmune response by the subject to the antigen polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C: Analysis of modified Rep78 constructs. a) Comparison ofcalculated codon pair bias scores of wild-type Rep78, Scrambled Rep 78and Deoptimized Rep78. b) Western blot for expression levels fromflag-tagged wtRep78, Scr Rep78 and Deopt Rep78 ORFs, expressed under aCMV promoter. Levels of GAPDH serve as loading controls. c) Densitometryanalysis of Western blot bands for quantification of expression levelswas performed using Gel Pro Analyzer 3.0. Levels of Rep expression werenormalized to GAPDH levels.

FIG. 2A-C: Functional analyses of genetically recoded Rep 78. (A) Schemaof genetic constructs, viruses, and predicted monomeric and dimericforms resulting from productive Rep 78 cleavage of Ad/AAV/PF4/BDD [basedon previously-characterized models [Gnatenko, 2004 #1359; Sandalon, 2000#1379]]. ψ—adenoviral packaging sequence; pCMV—cytomegalovirus corepromoter; Tet—Tetracycline response element/transactivator;pTK—thymidine kinase promoter; IRES-EGFP—internal ribosome entry sitewith enhanced GFP; Ad—adenovirus bp 3390-3940; BDD—Human B-domaindeleted factor VIII (24); pPF4—platelet factor 4 promoter (7); p5IEE(135 bp) and AAV TR (145 bp each plus G-C tail) are also shown. (B)Excision assays were completed by transfecting 293 cells with individualpAd/Rep plasmids, followed by Ad/AAV/PF4/BDD infection (MOI 50), in thepresence (+) or absence (−) of doxycycline (1 μg/mL) for evaluation ofdimeric (D) or monomeric (M) Ad/AAV/PF4/BDD excision products, generatedonly with functional Rep 78 endonuclease cleavage at the right TR (9).Southern blots were completed loading 1 μg Hirt DNA/lane, using the ˜800bp DIG labeled PF4/BDD junction fragment as probe. pIM45 is an AAVplasmid expressing wild-type Rep and Cap genes, used as positive controlfor efficient excision. Faint, low-level excision in the absence ofdoxycycline is presumably due to “leaky” Rep 78 expression. (C) Excisionassays using viral co-infections (MOI 50) were completed in 293 or C12cells using Ad/sRep or Ad/dRep and Ad/AAV/PF4/BDD, in the presence ofdox, 48 hours prior Hirt DNA isolation and Southern blot analysis asoutlined above. C12 cells are HeLa-derived stable cell lines thatconstitutively provide Rep and Cap, used as positive controls. In panels(FIG. 2B) and (FIG. 2C), parent Ad/AAV/PF4/BDD virus is depicted by anasterisk.

FIG. 3A-C: Computational prediction model for Rep inhibitory sequence:(A) Chimeric Rep genes were assembled by polynucleotide domain swapsencompassing discrete ˜600 bp segments of wild-type or scrambledsequences, and viability established by adenoviral replication andtitering assays in HEK 293 packaging cells (9, 24). Numbers above theschema denote the positions where the chimeric Rep genes were assembled.+/− to the right indicate viability of the resultant adenoviruses. Referto Table 1 for detailed viral titers. (B) Four distinct Rep chimers eachcontaining 14 discrete (132-135 bp) segments of wild-type or scrambledsequences were synthesized, and Ad replication (viability) was studiedin HEK 293 cells (refer to Supplement for nucleotide sequences). Each ofthe 14 segments has distinct patterns of wild-type or scrambled Repsequences. Thus the pattern of observed viability uniquely determinesthe location of the critical signal. Details of the design are describedin Skiena & Ward. Note that the columns can be permuted in any of 14!(˜8.7×10¹⁰) orderings with equivalent ability to identify criticalsequences, provided it lies completely within one of 14 segments. Tominimize the effect of signals on boundaries, columns were ordered tominimize transitions, in effect creating a balanced Gray (binary) codewhose distinct genetic signatures and phenotypic growth patterns can beapplied for delineation of critical Rep inhibitory sequences (in thiscase delineated by the *, encompassing Rep sequences bp 1782-1918). (C)Southern blot analysis using Hirt (episomal) DNA isolated at Day 2 orDay 10 post-transfection with the four constructs depicted in FIG. 3B(Ad/Rep I, Ad/Rep II, Ad/Rep II, Ad/Rep IV) with the DNA double digestedwith DpnI/SbfI and the Southern blot probed using DIG labeled pTG3602ΔE3 F5/35 DNA.

FIG. 4A-H: (A) Wild type AAV Rep inhibitory nucleotide sequence (SEQ IDNO:01), 135-bp, from AAV2 genome (GenBank accession number AF043303.1)bp 1782 to bp 1916, (B) Wild type AAV Rep inhibitory polypeptidesequence (SEQ ID NO:02) encoded by SEQ ID NO:01, (C) Scrambled AAV Repinhibitory nucleotide sequence (SEQ ID NO:07), which corresponds to the135-bp wild type AAV Rep inhibitory nucleotide sequence (SEQ ID NO:01),(D) Deoptimized AAV Rep inhibitory nucleotide sequence (SEQ ID NO:09),which corresponds to the 135-bp wild type AAV Rep inhibitory nucleotidesequence (SEQ ID NO:01), (E) Wild type AAV Rep inhibitory nucleotidesequence SEQ ID NO:17 (564-bp sequence from bp 1623 to bp 2186, ofAdeno-Associated Virus 2 (AAV2) genome GenBank: AF043303.1. The 135-bpwild type AAV Rep inhibitory nucleotide sequence (SEQ ID NO:01) isunderlined. (F) Wild type AAV Rep inhibitory polypeptide sequence (SEQID NO:20) encoded by SEQ ID NO: 17, (G) Scrambled AAV Rep inhibitorynucleotide sequence (SEQ ID NO:18), which corresponds to the 564-bp wildtype AAV Rep inhibitory nucleotide sequence SEQ ID NO:17, and (H)Deoptimized AAV Rep inhibitory nucleotide sequence (SEQ ID NO:19), whichcorresponds to the 564-bp wild type AAV Rep inhibitory nucleotidesequence SEQ ID NO:17.

FIG. 5: Nucleotide sequence (SEQ ID NO:03) encoding an exemplary wildtype AAV2 Rep78. A portion of the Rep68 sequence lies within the Rep78sequence from bp 321 to bp 1906 of the AAV2 genome). The 3′ end theRep68 sequence lies downstream of the Rep78 stop codon. The sequencecommon to Rep68 and Rep78 is underlined. The inhibitory sequence (bp1782 to bp 1916 of the AAV2 genome). The nucleotide sequence (SEQ IDNO:01) of the exemplary 135-bp wild type AAV Rep inhibitory nucleotidesequence, from bp 1782 to bp 1916 of the AAV2 genome, is in italics.

FIG. 6A-B: (A) Amino acid sequence (SEQ ID NO:04) of AAV wild type Rep78protein (GenBank protein_id=“AAC03775.1, db_xref=“GI:2906018. (B) Aminoacid sequence (SEQ ID NO:05) of AAV wild type Rep68 protein (GenBankprotein_id=“AAC03774.1, db_xref=“GI:2906017.

FIG. 7A-B: (A) Scrambled AAV Rep78 nucleotide sequence sRep78 (SEQ IDNO:06). Sequence of the exemplary scrambled AAV Rep inhibitorynucleotide sequence (SEQ ID NO:07), which corresponds to the 135-bp wildtype AAV Rep inhibitory nucleotide sequence (SEQ ID NO:01) is initalics. (B) Deoptimized AAV Rep78 nucleotide sequence dRep78 (SEQ IDNO:08). Sequence of the exemplary deoptimized AAV Rep inhibitorynucleotide sequence (SEQ ID NO:09), which corresponds to the 135-bp wildtype AAV Rep inhibitory nucleotide sequence (SEQ ID NO:01) is initalics.

FIG. 8: Genome organization of AAV (Merten-O-W et al. 2005:12:S51-61).

FIG. 9: Adenovirus genome (Alba et al. Gene Therapy 2005:12:S18-27).

FIG. 10A-F: Alignments of sRep78 (S) (SEQ ID NO:06) and dRep78 (D) (SEQID NO:08) with wtRep78 (W) (SEQ ID NO:03).

FIG. 11: Schematic representation of the 138-nt IEE (SEQ ID NO:11)showing YY1 and Rep-binding sites, a putative upstream stimulatingfactor (USF)-binding site, and a TATA box (Philpott et al. (2002) A p5integration efficiency element mediates Rep-dependent integration intoAAVS1 at chromosome 19. Proc. Natl. Acad. Sci. USA 99:12381).

FIG. 12A-C: Nucleotide sequence (SEQ ID NO:16) of the wild type completegenome of Adeno-Associated Virus 2 (AAV2), GenBank: AF043303.1,containing wild type AAV Rep inhibitory nucleotide sequence SEQ ID NO:17(564-bp sequence from bp 1623 to bp 2186, shown in underlined boldtext), which in turn contains wild type AAV Rep inhibitory nucleotidesequence SEQ ID NO:01 (135-bp sequence from bp 1782 to bp 1916, shown inunderlined bold, italicized text).

FIG. 13A-C: Alignments of wild-type (WT), deoptimized (d), and scrambled(s) AAV Rep. Nucleotides identical to all three sequences are delineatedby *. Alignments of the 1,866 base pair re-coded Rep genomic sequenceswere completed and displayed using Clustal 2.1 multiple sequencealignment tool, numbered relative to the full-length 4,679 bp AAV2genome (Gen Bank Accession number AF043303.1, SEQ ID NO: 16 of FIG. 12A-C); the circle (•) delineates the initiator MET starting at bp 321;the p19 (bp 843 to 849) and p40 (bp 1,823 to 1,827) promoter TATAsequences, and the 5′-Rep 68/40 alternative splice site (bp 1,907 to1,908) are highlighted in a grey box. The wild type AAV Rep inhibitorynucleotide sequence SEQ ID NO:17 (from bp 1623 to bp 2186) is shown inunderlined bold text, and contains the wild type AAV Rep inhibitorynucleotide sequence SEQ ID NO:01 (135-bp long, from bp 1782 to bp 1916,shown in underlined bold, italicized text). The previously-characterizedp40 promoter transcription binding sites for GGT, SP1, and API areboxed; the transcription start site is delineated by an arrow.

FIG. 14A-B. Additional delineation of Rep inhibitory sequences. ChimericRep genes were assembled by polynucleotide segment swaps encompassingdiscrete segments of wild-type or scrambled sequences, and viabilityestablished by adenoviral replication. (A) Rep78 chimer encompassingwild-type 135 bp sequences corresponding to segment 11 and 12 on thebackground of sRep. (B) Rep78 chimer encompassing wild-type 135 bpsequences corresponding to segment 12 and 13 on the background of sRep.

FIG. 15A-D. Rep78 nucleotide and amino acid sequences. A. Segment 11: bp1647-1781 of GenBank accession number AF043303.1: Wild type nucleotidesequence (SEQ ID NO:21), Wild type amino acid sequence (SEQ ID NO:22),Scrambled nucleotide sequence (SEQ ID NO:23). B. Segment 13: bp1917-2051 of GenBank accession number AF043303.1: Wild type nucleotidesequence (SEQ ID NO:24), Wild type amino acid sequence (SEQ ID NO:25),Scrambled nucleotide sequence (SEQ ID NO:26). C. Segment 14: bp2052-2186 of GenBank accession number AF043303.1: Wild type nucleotidesequence (SEQ ID NO:27), Wild type amino acid sequence (SEQ ID NO:28),Scrambled nucleotide sequence (SEQ ID NO:29). D. Segment 12: 135 bpsequence: bp1782-1916 of GenBank accession number AF043303.1: Wild typenucleotide sequence (SEQ ID NO:01), Wild type amino acid sequence (SEQID NO:02), Scrambled nucleotide sequence (SEQ ID NO:07).

FIG. 16A-D. Polynucleotide sequences encoding wild type AAV Repinhibitory amino acid sequence. A. Segment 11 scrambled nucleotidesequence (SEQ ID NO:23). B. Segment 13 scrambled nucleotide sequence(SEQ ID NO:26). C. Segment 14 scrambled nucleotide sequence (SEQ IDNO:29). D. Segment 12 scrambled 135 bp nucleotide sequence (SEQ IDNO:07).

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below. Further definitions appear throughout the text.

The term “recombinant” nucleotide sequence refers to a nucleotidesequence that is produced by means of molecular biological techniques(e.g., cloning, enzyme restriction and/or ligation steps) and/orchemical synthesis.

“Recombinant protein” or “recombinant polypeptide” refers to a proteinmolecule that is expressed using a recombinant nucleotide sequence.

“Recombinant mutation” refers to a mutation that is introduced by meansof molecular biological techniques. This is in contrast to mutationsthat occur in nature.

“Recombinant virus” refers to a virus that contains a recombinantnucleotide sequence, recombinant polypeptide, and/or recombinantmutation, as well as progeny of that virus.

“Endogenous,” “wild type,” “wildtype,” “wt” and “wild-type” when inreference to a sequence (e.g., that is introduced into a cell and/orvirus) refer to the sequence as it occurs in nature (e.g., in the celland/or virus). It is now appreciated that most or all gene loci exist ina variety of allelic forms, which vary in frequency throughout thegeographic range of a species. Thus, in one embodiment, a “wild type”sequence is the sequence that occurs at the highest frequency in nature.

The term “heterologous” when in reference to a sequence (e.g., that isintroduced into a cell and/or virus) refers to a sequence that is notendogenous (to the cell and/or virus into which it is introduced). Forexample, a “heterologous” gene refers to a gene that is not in itsnatural environment (in other words, has been altered by the hand ofman). For example, a heterologous gene includes a gene from one speciesintroduced into another species. A heterologous gene also includes agene native to an organism that has been altered in some way (forexample, mutated, added in multiple copies, linked to a non-nativepromoter or enhancer sequence, etc.). Heterologous genes may comprisecDNA forms of a gene; the cDNA sequences may be expressed in either asense (to produce mRNA) or anti-sense orientation (to produce ananti-sense RNA transcript that is complementary to the mRNA transcript).Heterologous genes are distinguished from endogenous genes in that theheterologous gene sequences are typically joined to nucleotide sequencescomprising regulatory elements such as promoters that are not foundnaturally associated with the gene for the protein encoded by theheterologous gene or with gene sequences in the chromosome, or areassociated with portions of the chromosome not found in nature (forexample, genes expressed in loci where the gene is not normallyexpressed).

The term “operably linked” when in reference to the relationship betweennucleic acid sequences and/or amino acid sequences refers to linking thesequences such that they perform their intended function. For example,operably linking a promoter sequence to a nucleotide sequence ofinterest refers to linking the promoter sequence and the nucleotidesequence of interest in a manner such that the promoter sequence iscapable of directing the transcription of the nucleotide sequence ofinterest and/or the synthesis of a polypeptide encoded by the nucleotidesequence of interest. Similarly, operably linking AAV terminal repeats(TRs) to a nucleotide sequence of interest means that the sequences arelinked in such a way such that the AAV TRs are capable of directingreplication of the nucleotide sequence of interest. Also, operablylinking an AAV packaging sequence to a nucleotide sequence of interestrefers to linkage of these sequences such that the AAV packagingsequence is capable of directing packaging of the nucleotide sequence ofinterest into an encapsidated virion.

“Portion” and “fragment” when made in reference to a nucleic acidsequence or protein sequence refer to a piece of that sequence that mayrange in size from two (2) contiguous nucleotides and amino acids,respectively, to the entire sequence minus one nucleotide and aminoacid, respectively. Thus. “at least a portion of” a nucleic acidsequence or protein sequence refers to a piece of that sequence that mayrange in size from two (2) contiguous nucleotides and amino acids,respectively, to the entire sequence. For example, “at least a portionof the 135-base pair wild type AAV Rep inhibitory amino acid sequencelisted as SEQ ID NO:02” refers to a sequence that ranges in size fromany numerical value from 2 to 135 contiguous base-pairs, such as 2, 3,4, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,and 135 contiguous base-pairs. Similarly, “at least a portion of the564-base pair wild type AAV Rep inhibitory amino acid sequence listed asSEQ ID NO:20” refers to a sequence that ranges in size from anynumerical value from 2 to 564 contiguous base-pairs, such as from 2 to50, from 2 to 100, from 2 to 150, from 2 to 200, from 2 to 200, from 2to 250, from 2 to 300, from 2 to 350, from 2 to 400, from 2 to 450, from2 to 500, and from 2 to 564 contiguous base-pairs.

A “functional” portion of a sequence (e.g., polypeptide orpolynucleotide sequence) refers to a portion of the sequence that hasone or more activities (e.g., enzyme activity, biochemical activity,etc.) of the full-length sequence. For example, a functional portion ofa promoter refers to a nucleic acid sequence that is capable of bindingto RNA polymerase to initiate transcription of an operably linkedoligonucleotide sequence into mRNA. In another example, a functionalportion of Rep78 protein refers to a portion of Rep78 that functions incleaving a folded AAV ITR. Methods for determining this function areknown in the art and described herein (Example 3).

“Chimeric,” “fusion” and “hybrid” composition (e.g., when in referenceto an amino acid sequence, nucleotide sequence, virus, cell, etc.)refers to a composition containing parts from different origins. In oneembodiment, the parts may be from different organisms, differenttissues, different cells, different viruses, etc. In another embodiment,the parts may be from different proteins and/or genomic sequences fromthe same organism, same tissue, same cell, same virus, etc.

The terms “mutation” and “modification” refer to a deletion, insertion,or substitution. A “deletion” is defined as a change in a nucleic acidsequence or amino acid sequence in which one or more nucleotides oramino acids, respectively, is absent. An “insertion” or “addition” isthat change in a nucleic acid sequence or amino acid sequence that hasresulted in the addition of one or more nucleotides or amino acids,respectively. A “substitution” in a nucleic acid sequence or an aminoacid sequence results from the replacement of one or more nucleotides oramino acids, respectively, by a molecule that is a different moleculefrom the replaced one or more nucleotides or amino acids. For example, anucleic acid may be replaced by a different nucleic acid as exemplifiedby replacement of a thymine by a cytosine, adenine, guanine, or uridine.Alternatively, a nucleic acid may be replaced by a modified nucleic acidas exemplified by replacement of a thymine by thymine glycol.Substitution of an amino acid may be conservative or non-conservative.“Conservative substitution” of an amino acid refers to the replacementof that amino acid with another amino acid that has a similarhydrophobicity, polarity, and/or structure. For example, the followingaliphatic amino acids with neutral side chains may be conservativelysubstituted one for the other: glycine, alanine, valine, leucine,isoleucine, serine, and threonine. Aromatic amino acids with neutralside chains that may be conservatively substituted one for the otherinclude phenylalanine, tyrosine, and tryptophan. Cysteine and methionineare sulphur-containing amino acids that may be conservativelysubstituted one for the other. Also, asparagine may be conservativelysubstituted for glutamine, and vice versa, since both amino acids areamides of dicarboxylic amino acids. In addition, aspartic acid(aspartate) may be conservatively substituted for glutamic acid(glutamate) as both are acidic, charged (hydrophilic) amino acids. Also,lysine, arginine, and histidine may be conservatively substituted onefor the other since each is a basic, charged (hydrophilic) amino acid.“Non-conservative substitution” is a substitution other than aconservative substitution. Guidance in determining which and how manyamino acid residues may be substituted, inserted or deleted withoutabolishing biological and/or immunological activity may be found usingcomputer programs well known in the art, for example, DNAStar™ software.

A “variant” or “homolog” of a polypeptide sequence of interest ornucleotide sequence of interest refers to a sequence that has identityof at least 65% with the an amino acid sequence of interest ornucleotide sequence of interest, including identity of at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, and/or at least 99%. Thus,homologous genomic nucleotide sequences within the scope of theinvention include orthologs and paralogs. The term “ortholog” refers toa gene in different species that evolved from a common ancestral gene byspeciation. In some embodiments, orthologs retain the same function. Theterm “paralog” refers to genes related by duplication within a genome.In some embodiments, paralogs evolve new functions. In furtherembodiments, a new function of a paralog is related to the originalfunction. Variants of a polypeptide sequence of interest may contain amutation.

“Identity” when in reference to 2 or more sequences (e.g., 2 DNAsequences, 2 RNA sequences, and/or 2 protein sequences) refers to thedegree of similarity of the 2 or more sequences, and is generallyexpressed as a percentage. Identity in amino acid or nucleotidesequences can be determined using Karlin and Altschul's BLAST algorithm(Proc. Natl. Acad. Sci. USA, 1990, 87, 2264-2268; Karlin, S. & Altschul,S F., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873). Programs calledBLASTN and BLASTX have been developed using the BLAST algorithm as abase (Altschul, S F. et al., J. Mol. Biol., 1990, 215, 403). When usingBLASTN to analyze nucleotide sequences, the parameters can be set at,for example, score=100 and word length=12. In addition, when usingBLASTX to analyze amino acid sequences, the parameters can be set at,for example, score=50 and word length=3. When using BLAST and the GappedBLAST program, the default parameters for each program are used.Specific techniques for these analysis methods are the well known, e.g.,on the website of the National Center for Biotechnology Information.

The term “corresponding” when in reference to the position of a firstamino acid in a first polypeptide sequence as compared to a second aminoacid in a second polypeptide sequence means that the positions of thefirst and second amino acids are aligned when the first and second aminoacid sequences are aligned. Software for alignment of amino acidsequences and of nucleotide sequences is known in the art such as BLAST,FASTA, HMMER, IDF, SAM, and SSEARCH (for both amino acid sequences andnucleotide sequences), Infernal (for RNA sequences), CS-BLAST, HHpred,HHsearch, and PSI-BLAST (for amino acid sequences).

“Codon” refers to a specific sequence of three adjacent nucleotides on astrand of DNA or RNA that specifies the genetic code information forsynthesizing a particular amino acid.

“Codon pair” and “codon-pair” interchangeably refer to two codons thatare separated by less than 6 intervening nucleotides, i.e., separated byless than two intervening codons.

“Synonymous codon” when used to describe a first codon as compared to areference codon, refers to a first codon that differs in nucleotidesequence from the reference codon and that encodes the same amino acid.Due to the degeneracy of the codon table, 18 of the 20 amino acids canbe encoded using more than one codon. Synonymous codons differ from oneanother often at the third base of the codon (the wobble position). Thusthe same polypeptide sequence can be encoded by different nucleotidesequences that vary from one another by an amount equal to or less than33% (i.e., the nucleotide sequences have more than 67% identity).

“Synonymous codon pair” when used to describe a first codon pair ascompared to a reference codon pair, refers to a first codon paircontaining at least one synonymous codon compared to the correspondingcodon in the reference codon pair. Thus, in one embodiment, a synonymouscodon pair contains one synonymous codon compared to the correspondingcodon in the reference codon pair. In another embodiment, both codons inthe synonymous codon pair are synonymous codons compared to thecorresponding codons in the reference codon pair.

“Codon bias’ refers to the presence of a different (higher or lower)frequency of using one synonymous codon than another synonymous codon toencode the same amino acid. For instance, in humans, the Ala codon GCCis used four times as frequently as the synonymous codon GCG (Coleman etal. (2008)). An “underrepresented codon” refers to a codon that occursat a lower frequency compared with random frequency for that codon. Incontrast, an “overrepresented codon” refers to a codon that occurs at ahigher frequency compared with random frequency for that codon.

“Codon pair bias” refers to the presence of a different (higher orlower) frequency of using a codon-pair compared with random frequencyfor that codon-pair (Gutman et al. (1989)). Thus, in one embodiment,codon pair bias refers to the preference (i.e., (higher or lowerfrequency) for some codon pairs over other synonymous codons to encodethe same pair of adjacent amino acids. Synonymous codons can be pairedin multiple ways to encode the same 2 adjacent amino acids. However, innature a strong codon pair bias is found to exist, resulting in thedisproportionate representation of some codon pairs over others (Gutmanet al. (1989) Nonrandom utilization of codon pairs in Escherichia coli.Proc. Natl. Acad. Sci. U.S.A. 86(10):3699-3703). This codon pair bias isindependent of codon frequency and is found to affect translation rates.Codon pair bias includes under-representation of a codon pair, andoverrepresentation of a codon pair. An “underrepresented codon pair”refers to a codon pair that occurs at a lower frequency compared withrandom frequency for that codon-pair. In contrast, an “overrepresentedcodon pair” refers to a codon pair that occurs at a higher frequencycompared with random frequency for that codon-pair. For example, theamino acid pair Ala-Glu is expected to be randomly encoded by the codonpair GCCGAA and the codon pair GCAGAG about equally often. However, thecodon pair GCCGAA is underrepresented such that it is used onlyone-seventh as often as the codon pair GCAGAG.

“Scrambled nucleotide sequence” and “Scr nucleotide sequence” whendescribing a first nucleotide sequence as compared to a referencenucleotide sequence (e.g., a reference wild type AAV Rep inhibitorynucleotide sequence listed as SEQ ID NO:17 or SEQ ID NO:01)interchangeably refer to a first nucleotide sequence that contains oneor more synonymous codons and/or one or more synonymous codon-pairs, andthat encodes the same amino acid sequence that is encoded by thereference nucleotide sequence. The prior art provides algorithm forscrambling nucleotide sequences using synonymous codons without alteringthe encoded amino acid sequence (Coleman et al. (2008) Science320(5884):1784-1787) (Example 2). Thus, a “codon-scrambled nucleotidesequence” refers to a first nucleotide sequence that contains one ormore synonymous codons compared to a reference nucleotide sequence, andthat encodes the same amino acid sequence that is encoded by thereference nucleotide sequence. A “codon-pair-scrambled nucleotidesequence” refers to a first nucleotide sequence that contains one ormore synonymous codon-pairs compared to a reference nucleotide sequence,and that encodes the same amino acid sequence that is encoded by thereference nucleotide sequence. In one embodiment, the synonymous codonsand/or codon-pairs are randomly mixed. In another embodiment, from 50%to 100% of the codons and/or codon-pairs of a scrambled nucleotidesequence is synonymous to the codons and/or codon-pairs, respectively,of the reference sequence, including, for example, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, and 100% synonymous codons and/or synonymouscodon-pairs, respectively. In one preferred embodiment, 100% of thecodons and/or codon-pairs of a scrambled nucleotide sequence issynonymous to the codons and/or codon-pairs, respectively, of thereference sequence. Thus, in one embodiment in which 100% of the codonsand/or 100% of the codon-pairs of a scrambled nucleotide sequence issynonymous to the codons and/or codon-pairs of a reference sequence,respectively, then the scrambled nucleotide sequence differs from thereference sequence by an amount equal to or less than 33%, i.e., thescrambled nucleotide sequence has more than 67% identity to thereference sequence. For example, the invention provides a “scrambled AAVRep inhibitory nucleotide sequence listed as SEQ ID NO:18 (FIG. 4G)and/or SEQ ID NO:07” (FIG. 4, C, and FIG. 7, A) also referred to as a“scrambled polynucleotide sequence encoding wild type AAV Rep inhibitoryamino acid sequence listed as SEQ ID NO:20 (FIG. 4F) and/or SEQ IDNO:02,” (FIG. 4 B) wherein the reference nucleotide sequence is the wildtype AAV Rep inhibitory nucleotide sequence listed as SEQ ID NO:17 (FIG.4E, FIG. 12A-C, FIG. 13A-C and/or SEQ ID NO:01 (FIG. 4 A, FIG. 12A-C,FIG. 13A-C). In one embodiment, the level of protein expression by thescrambled nucleotide sequence is substantially the same as the level ofexpression by the reference nucleotide sequence and/or is approximatelytwice the level of expression by a deoptimized nucleotide sequence (Asillustrated, without limitation in Example 2). Generic methods (e.g.,algorithms) for generating a scrambled nucleotide sequence by modifyinga reference nucleotide sequence without modifying the encoded amino acidsequence are know in the art (Coleman J R, et al. (2008).

A “deoptimized nucleotide sequence” when describing a first nucleotidesequence as compared to a reference nucleotide sequence (e.g., areference wild type AAV Rep inhibitory nucleotide sequence listed as SEQID NO:17 or SEQ ID NO:01) means a scrambled first nucleotide sequencethat contains one or more underrepresented codons and/or one or moreunderrepresented codon-pairs (see for Example FIG. 1a ), and thatencodes the same amino acid sequence that is encoded by the referencenucleotide sequence. Thus, a “codon-deoptimized nucleotide sequence”refers to a first nucleotide sequence that contains one or moredeoptimized codons compared to a reference nucleotide sequence, and thatencodes the same amino acid sequence that is encoded by the referencenucleotide sequence. A “codon-pair-deoptimized nucleotide sequence”refers to a first nucleotide sequence that contains one or moredeoptimized codon-pairs compared to a reference nucleotide sequence, andthat encodes the same amino acid sequence that is encoded by thereference nucleotide sequence. In a further embodiment, from 50% to 100%of the codons and/or codon-pairs of a deoptimized nucleotide sequence isdeoptimized as compared to the codons and/or codon-pairs, respectively,of the reference sequence, including, for example, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, and 100% deoptimized codons and/or deoptimizedcodon-pairs, respectively. In one embodiment, 100% of the codons and/orcodon-pairs of a deoptimized nucleotide sequence are underrepresentedcodons and/or codon-pairs compared to the codons and/or codon pairs,respectively, of the reference sequence. In one embodiment, utilizationof underrepresented codons and/or codon pairs in a deoptimizednucleotide sequence results in a lower level of expression of thedeoptimized nucleotide sequence compared to the level of expression of ascrambled nucleotide sequence and/or of a control wild type sequence,due to inefficient translation. For example, data herein show that thelevel of Rep78 protein expression by the scrambled Rep78 nucleotidesequence is substantially [OK defined] the same as the level ofexpression by the reference wild type Rep78 nucleotide sequence, andapproximately twice the level of expression by a deoptimized Rep78nucleotide sequence (see Example 2, FIG. 1b ). Confirmation of thereduced protein expression by a deoptimized nucleotide sequence may bedetermined using methods known in the art (e.g., immunoblot analysis,Example 2). In one embodiment, the invention provides a “deoptimized AAVRep inhibitory nucleotide sequence” that comprises SEQ ID NO:19 (FIG.4H) and/or SEQ ID NO:09 (FIG. 4 D, FIG. 7 B), and that encodes wild typeAAV Rep inhibitory amino acid sequence listed as SEQ ID NO:20 (FIG. 4F)and/or SEQ ID NO:02 (FIG. 4 B).

The terms “lack” and “lacking” a nucleotide sequence when made inreference to a vector means that the vector contains at least onedeletion (i.e., absence of one or more nucleotides) in the nucleotidesequence. Deletions may be continuous (i.e., uninterrupted) ordiscontinuous (i.e., interrupted). Deletions may lie in a codingsequence or a regulatory sequence. A deletion can be a partial deletion(i.e., involving removal of a portion ranging in size from one (1)nucleotide residue to the entire nucleic acid sequence minus one nucleicacid residue) or a total deletion of the nucleotide sequence. Deletionsare preferred which prevent the production of at least one expressionproduct encoded by the nucleotide sequence. For example, a vector thatlacks adenovirus E1 gene region refers to a vector that contains atleast one deletion in the E1 gene region. Preferably, though notnecessarily, the deletion prevents the production of at least one of themultiple proteins encoded by the E1 gene region.

“Virus” refers to an obligate, ultramicroscopic, intracellular parasiteparticle of nucleic acid sequence (DNA or RNA) that is assembled insidea polypeptide shell, and that is incapable of autonomous replication(i.e., replication requires the use of a host cell's machinery).

“Helper virus” refers to a virus that is replication-competent and/orproductive and/or infectious in a particular host cell (e.g., the hostcell may provide virus gene products such as adenovirus E1 proteins fora helper adenovirus that is replication-competent). For example, areplication-competent and/or productive and/or infectious first virus(i.e., helper virus) is used to supply, in trans, functions (e.g.,proteins) that are lacking in a second virus that isreplication-incompetent and/or non-productive and/or non-infectious.Thus, the first virus is the to “help” the second virus therebypermitting the replication by and/or production of and/or infection bythe second viral genome in the cell containing both the first helpervirus and the second viruses.

The terms “free of helper virus” and “free of contamination with helpervirus” when in reference to a sample, mean that the number of helpervirus particles in the sample is from zero % to 1%, more preferably fromzero % to 0.5%, and most preferably from zero % to 0.05%, when comparedto the number of particles of a second virus in the same sample.

The term “replication” of a virus includes, but is not limited to, thesteps of adsorbing (e.g., receptor binding) to a cell, entry into a cell(such as by endocytosis), introducing its genome sequence into the cell,un-coating the viral genome, initiating transcription of viral genomicsequences, directing expression of viral encapsidation proteins,encapsidating of the replicated viral nucleic acid sequence with theencapsidation proteins into a viral particle that is released from thecell to infect other cells that are of a permissive and/or susceptiblecharacter. A virus may be infectious (i.e., can penetrate a cell)without being replication competent (i.e., fails to release virions fromthe infected cell).

“Replication competent” when in reference to a viral vector and/or virusmeans capable of adsorbing (e.g., receptor binding) to a cell, entryinto a cell (such as by endocytosis), introducing its genome sequenceinto the cell, un-coating the viral genome, initialing transcription ofviral genomic sequences, directing expression of viral encapsidationproteins, encapsidating of the replicated viral nucleic acid sequencewith the encapsidation proteins into new progeny virus particles.

“Replication incompetent,” “replication defective,” “replicationattenuated” are used interchangeably to refer to a virus and/or viralvector that has a reduced level of replication compared to wild typevirus and/or to a viral vector containing wild type virus nucleotidesequences. Replication incompetent also means a virus particle that issubstantially incapable of completing one or more of the steps ofreplication. Methods for producing replication incompetent adenoviralvectors are known in the art (e.g., U.S. Pat. No. 7,300,657 to Pau, U.S.Pat. No. 7,468,181 to Vogels, U.S. Pat. No. 6,136,594 to Dalemans, U.S.Pat. No. 5,994,132 to Chamberlain et al., U.S. Pat. No. 6,797,265 toAmalfitano et al., U.S. Pat. No. 7,563,617 to Hearing et al., and U.S.Pat. No. 6,262,035 to Campbell et al.). For example, in one embodiment,a replication incompetent adenovirus and/or adenoviral vector (a) lacks(i.e., has a deletion of) adenovirus E1 gene coding sequence, (b) lacksadenovirus E1 gene coding sequence and E2b gene coding sequence (c)lacks adenovirus E1 gene coding sequence and adenovirus E4 gene codingsequence, (d) lacks adenovirus E1 gene coding sequence and adenovirusE2a gene coding sequence, and/or (e) lacks adenovirus E1 gene codingsequence and adenovirus EIVa2 gene coding sequence.

“Infection” and “infectious” when in reference to a virus refer toadsorption of the virus to the cell and penetration into the cell. Avirus may be infectious (i.e., can adsorb to and penetrate a cell)without being replication competent (i.e., fails to produce new progenyvirus particles). Data herein demonstrate productive infection thatgenerated infectious virus that is replication competent, using clonepAd/sRep78 (containing a scrambled Rep78 sequence) and clone pAd/dRep78(containing a deoptimized Rep78 sequence), that formed CPE intransfected HEK 293 packaging cells (Example 3).

A “non-infectious” and “uninfectious” virus is a virus that is incapableof adsorption to, and/or penetration into, a cell.

“Productive” virus is a replication competent virus that is capable of a“productive infection,” i.e., wherein the replication competent virusproduces new progeny virus particles that are released extracellularly.Productive infection by a productive virus may be detected by detectionof CPE. Data herein demonstrate productive infection that generatedinfectious virus that is replication competent, using clone pAd/sRep78(containing a scrambled Rep78 sequence (SEQ ID NO:06 of FIG. 7 pane A))and clone pAd/dRep78 (containing a deoptimized Rep78 sequence (SEQ IDNO:08 of FIG. 7 B)), that formed CPE in transfected HEK 293 packagingcells (Example 3).

“Non-productive” virus is a replication competent virus that produces a“non-productive infection,” i.e., wherein the replication competentvirus produces new progeny virus particles that are not released fromthe infected cell. This includes scenarios where the viral genome isintegrated into the host cell genome. Non-productive infection by anon-productive virus may be detected by detecting virus proteins and/ornucleic acids in cellular extracts, in the absence of CPE.

“Encapsidated” when made in reference to a nucleotide sequence refers toa nucleotide sequence that is packaged inside a protein envelope to forma particle. Data presented herein demonstrates that the invention'snucleotide sequence vectors were packaged efficiently into stable virusparticles. Encapsidated vectors of the invention may be recoveredfollowing transfection or infection of target cells using methods knownin the art. When used herein, “recovering” encapsidated vectors refersto the collection of the vectors by, for example, lysis of the cell(e.g., freeze-thawing) and removing the cell debris by pelleting, and/orcollection of extracellular solutions.

The terms “cytopathic effect” and “CPE” as used herein describe changesin cellular structure (i.e., a pathologic effect). Common cytopathiceffects include cell destruction, syncytia (i.e., fused giant cells)formation, cell rounding, vacuole formation, and formation of inclusionbodies. CPE results from actions of a virus on permissive cells thatnegatively affect the ability of the permissive cellular host to performits required functions to remain viable. In in vitro cell culturesystems, CPE is evident when cells, as part of a confluent monolayer,show regions of non-confluence after contact with a specimen thatcontains a virus. The observed microscopic effect is generally focal innature and the foci are initiated by a single virion. However, dependingupon viral load in the sample, CPE may be observed throughout themonolayer after a sufficient period of incubation. Cells demonstratingviral induced CPE usually change morphology to a rounded shape, and overa prolonged period of time can die and be released from their anchoragepoints in the monolayer. When many cells reach the point of focaldestruction, the area is called a viral plaque, which appears as a holein the monolayer. The terms “plaque” and “focus of viral infection”refer to a defined area of CPE which is usually the result of infectionof the cell monolayer with a single infectious virus which thenreplicates and spreads to adjacent cells of the monolayer. Cytopathiceffects are readily discernable and distinguishable by those skilled inthe art. Data herein demonstrate productive infection that generatedinfectious virus that is replication competent, using clone pAd/sRep78(containing a scrambled Rep78 sequence) and clone pAd/dRep78 (containinga deoptimized Rep78 sequence), that formed CPE in transfected HEK 293packaging cells (Example 3).

“Integration” of a first nucleotide sequence (e.g., a transgene) into asecond nucleotide sequence (e.g., a genome) refers to the insertion ofthe first nucleotide sequence at one or more locations (referred to as“integration sites”) within the second nucleotide sequence followingcontacting the first and second nucleotide sequences. “Efficiency ofintegration” refers to the number of inserted first nucleotide sequencesrelative to the number of first nucleotide sequences that were contactedwith the second nucleotide sequence. Methods for determining efficiencyof integration are known in the art (McCarty et al. (2004) Annual Reviewof Genetics 38:819-844), including quantitative real-time PCR assays(Huser et al. (2002) J. Virol. 76:7554).

“Site-specific integration” and “SSI” refer to the insertion of thefirst nucleotide sequence occurs at one or more particular locations(“integration sites”) in the second nucleotide sequence. In oneembodiment, site-specific integration of a transgene into chromosome 19AAVS1 sites may be effected by using Rep68/78 proteins in trans to thetransgene and an “Rep Binding Element” (“RBE”) in cis (Feng et al.(2006) A 16 bp Rep Binding Element is Sufficient for MediatingRep-dependent Integration into AAVS1. Journal of Molecular Biology:1-8).This RBE can be found either within the AAV Terminal Repeat or the p5Integration Efficiency Element (p5IEE). Thus, for example, site-specificintegration of a transgene into chromosome 19 AAVS1 sites may beeffected by using the AAV ITR or IEE in cis to the transgene, and theRep68/78 protein in trans Thus, in one particular embodiment,site-specific integration into the AAVS1 sites may be accomplished usingan AAV ITR flanked transgene and the Rep protein in trans (McLaughlin etal. (1988) Adeno-associated virus general transduction vectors: analysisof proviral structure. J. Virol. 62:1963-1973). In another embodiment,site-specific integration into the AAVS1 sites may be accomplished usingthe IEE in cis to the transgene, and the Rep protein in trans (Philpottet al. (2002) A p5 integration efficiency element mediates Rep-dependentintegration into AAVS1 at chromosome 19. Proc. Natl. Acad. Sci. USA99:12381).

“RSSSI” and “Rep mediated site-specific integration” interchangeablyrefer to site-specific integration that requires the activity of AAV Repprotein. Site-specific integration with wt AAV particles has been foundto be highly specific in multiple cell lines, with one study finding 94%of all AAV positive IB3-1 cells to have site-specific integration intoChromosome 19.

“AAVS1” and “adeno-associated virus integration site 1” sequenceinterchangeably refer to the well-characterized sequence on the q arm ofchromosome 19 (19q13.3qter). (Wang et al. (2006) Journal of Virology80(23):11699-11709; McLaughlin et al. (1990) Proc. Natl. Acad. Sci. USA87, 2211-2215; Samulski et al. (1991) EMBO J. 10, 3941-3950; Giraud etal. (1994) Proc. Natl. Acad. Sci. USA 91, 10039-10043; Kearns et al.(1996) Gene Ther. 3, 748-755).

“AAV p5IEE” and “AAV p5 integration efficiency element” refer to the AAVsequence that is active in cis to a transgene for bringing aboutsite-specific integration of the transgene into the chromosome 19 AAVS1sequence, in the presence of the AAV Rep68/78 protein in trans to thetransgene. “AAV p5IEE” is exemplified by the 138-nt IEE (SEQ ID NO:11)(FIG. 11) (Philpott et al. (2002) Proc. Natl. Acad. Sci. USA 99:12381).

“Gene therapy” refers to reducing one or more clinical and/orsub-clinical symptoms of disease in a subject by insertion of nucleotidesequences into the subject's cells to replace damaged or abnormal geneswith normal ones, and/or to provide new genetic instructions to helpfight disease. Viruses are used as gene delivery vectors, as exemplifiedby vectors using sequences from adenovirus, adeno-associated virus,herpes simplex virus, retrovirus, lentivirus, baculovirus, etc., asdescribed herein.

The term “control” as used herein when in reference to a sample (e.g.,cell, tissue, animal, virus, etc.) refers to any type of sample that oneof ordinary skill in the art may use for comparing to a test sample(e.g., cell, tissue, animal, virus, etc.) by maintaining the sameconditions in the control and test samples, except in one or moreparticular factors. In one embodiment, the comparison of the control andtest samples is used to infer a causal significance of this varied oneor more factors.

A “subject” that may benefit from the invention's methods includes anymulticellular animal, preferably a mammal. Mammalian subjects includehumans, non-human primates, murines, ovines, bovines, ruminants,lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.).Thus, mammalian subjects are exemplified by mouse, rat, guinea pig,hamster, ferret and chinchilla. The invention's compositions and methodsare also useful for a subject “in need of reducing one or more symptomsof” a disease includes a subject that exhibits and/or is at risk ofexhibiting one or more symptoms of the disease. For Example, subjectsmay be at risk based on family history, genetic factors, environmentalfactors, etc. This term includes animal models of the disease. Thus,administering a composition (which reduces a disease and/or whichreduces one or more symptoms of a disease) to a subject in need ofreducing the disease and/or of reducing one or more symptoms of thedisease includes prophylactic administration of the composition (i.e.,before the disease and/or one or more symptoms of the disease aredetectable) and/or therapeutic administration of the composition (i.e.,after the disease and/or one or more symptoms of the disease aredetectable). The invention's compositions and methods are also usefulfor a subject “at risk” for disease refers to a subject that ispredisposed to contracting and/or expressing one or more symptoms of thedisease. This predisposition may be genetic (e.g., a particular genetictendency to expressing one or more symptoms of the disease, such asheritable disorders, etc.), or due to other factors (e.g., environmentalconditions, exposures to detrimental compounds, including carcinogens,present in the environment, etc.). The term subject “at risk” includessubjects “suffering from disease,” i.e., a subject that is experiencingone or more symptoms of the disease. It is not intended that the presentinvention be limited to any particular signs or symptoms. Thus, it isintended that the present invention encompass subjects that areexperiencing any range of disease, from sub-clinical symptoms tofull-blown disease, wherein the subject exhibits at least one of theindicia (e.g., signs and symptoms) associated with the disease.

“Subject in need of reducing one or more symptoms of” a disease, e.g.,infection with a pathogen, includes a subject that exhibits and/or is atrisk of exhibiting one or more symptoms of the disease. For Example,subjects may be at risk based on family history, genetic factors,environmental factors, etc. This term includes animal models of thedisease.

The terms “pathogen” and “animal pathogen” refer to any organism whichcauses a disease in an animal. Pathogens include, but are not limitedto, viruses, bacteria, protozoa, nematodes, fungus, etc.

The terms “pathogenic” and “virulent” when in reference to amicroorganism, such as virus, bacteria, parasite, etc. refer to theability of the microorganism to produce an infectious disease in anotherorganism (e.g., mammal).

The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” andgrammatical equivalents (including “lower,” “smaller,” etc.) when inreference to the level of any molecule (e.g., amino acid sequence, andnucleic acid sequence, antibody, etc.), cell, virus, and/or phenomenon(e.g., expression, transcription, translation, viral infection, viralproductive infection, viral replication, viral replication competence,Rep-mediated nuclease activity, site-specific integration into a genome,helicase activity, disease symptom, binding to a molecule, specificityof binding of two molecules, affinity of binding of two molecules,specificity to disease, sensitivity to disease, affinity of binding,enzyme activity, etc.) in a first sample (or in a first subject)relative to a second sample (or relative to a second subject), mean thatthe quantity of molecule, cell and/or phenomenon in the first sample (orin the first subject) is lower than in the second sample (or in thesecond subject) by any amount that is statistically significant usingany art-accepted statistical method of analysis. In one embodiment, thequantity of molecule, cell and/or phenomenon in the first sample (or inthe first subject) is at least 10% lower than, at least 25% lower than,at least 50% lower than, at least 75% lower than, and/or at least 90%lower than the quantity of the same molecule, cell and/or phenomenon inthe second sample (or in the second subject). In another embodiment, thequantity of molecule, cell, and/or phenomenon in the first sample (or inthe first subject) is lower by any numerical percentage from 5% to 100%,such as, but not limited to, from 10% to 100%, from 20% to 100%, from30% to 100%, from 40% to 100%, from 50% to 100%, from 60% to 100%, from70% to 100%, from 80% to 100%, and from 90% to 100% lower than thequantity of the same molecule, cell and/or phenomenon in the secondsample (or in the second subject). In one embodiment, the first subjectis exemplified by, but not limited to, a subject that has beenmanipulated using the invention's compositions and/or methods. In afurther embodiment, the second subject is exemplified by, but notlimited to, a subject that has not been manipulated using theinvention's compositions and/or methods. In an alternative embodiment,the second subject is exemplified by, but not limited to, a subject tothat has been manipulated, using the invention's compositions and/ormethods, at a different dosage and/or for a different duration and/orvia a different route of administration compared to the first subject.In one embodiment, the first and second subjects may be the sameindividual, such as where the effect of different regimens (e.g., ofdosages, duration, route of administration, etc.) of the invention'scompositions and/or methods is sought to be determined in oneindividual. In another embodiment, the first and second subjects may bedifferent individuals, such as when comparing the effect of theinvention's compositions and/or methods on one individual participatingin a clinical trial and another individual in a hospital.

The terms “increase,” “elevate,” “raise,” and grammatical equivalents(including “higher,” “greater,” etc.) when in reference to the level ofany molecule (e.g., amino acid sequence, and nucleic acid sequence,antibody, etc.), cell, virus, and/or phenomenon (e.g., expression,transcription, translation, viral infection, viral productive infection,viral replication, viral replication competence, Rep-mediated nucleaseactivity, site-specific integration into a genome, helicase activity,disease symptom, binding to a molecule, specificity of binding of twomolecules, affinity of binding of two molecules, specificity to disease,sensitivity to disease, affinity of binding, enzyme activity, etc.) in afirst sample (or in a first subject) relative to a second sample (orrelative to a second subject), mean that the quantity of the molecule,cell and/or phenomenon in the first sample (or in the first subject) ishigher than in the second sample (or in the second subject) by anyamount that is statistically significant using any art-acceptedstatistical method of analysis. In one embodiment, the quantity of themolecule, cell and/or phenomenon in the first sample (or in the firstsubject) is at least 10% greater than, at least 25% greater than, atleast 50% greater than, at least 75% greater than, and/or at least 90%greater than the quantity of the same molecule, cell and/or phenomenonin the second sample (or in the second subject). This includes, withoutlimitation, a quantity of molecule, cell, and/or phenomenon in the firstsample (or in the first subject) that is at least 10% greater than, atleast 15% greater than, at least 20% greater than, at least 25% greaterthan, at least 30% greater than, at least 35% greater than, at least 40%greater than, at least 45% greater than, at least 50% greater than, atleast 55% greater than, at least 60% greater than, at least 65% greaterthan, at least 70% greater than, at least 75% greater than, at least 80%greater than, at least 85% greater than, at least 90% greater than,and/or at least 95% greater than the quantity of the same molecule, celland/or phenomenon in the second sample (or in the second subject). Inone embodiment, the first subject is exemplified by, but not limited to,a subject that has been manipulated using the invention's compositionsand/or methods. In a further embodiment, the second subject isexemplified by, but not limited to, a subject that has not beenmanipulated using the invention's compositions and/or methods. In analternative embodiment, the second subject is exemplified by, but notlimited to, a subject to that has been manipulated, using theinvention's compositions and/or methods, at a different dosage and/orfor a different duration and/or via a different route of administrationcompared to the first subject. In one embodiment, the first and secondsubjects may be the same individual, such as where the effect ofdifferent regimens (e.g., of dosages, duration, route of administration,etc.) of the invention's compositions and/or methods is sought to bedetermined in one individual. In another embodiment, the first andsecond subjects may be different individuals, such as when comparing theeffect of the invention's compositions and/or methods on one individualparticipating in a clinical trial and another individual in a hospital.

The terms “alter” and “modify” when in reference to the level of anymolecule and/or phenomenon refer to an increase and/or decrease.

“Substantially the same” when in reference to the level of any molecule(e.g., amino acid sequence, and nucleic acid sequence, antibody, etc.),cell, virus, and/or phenomenon (e.g., expression, transcription,translation, viral infection, viral productive infection, viralreplication, viral replication competence, Rep-mediated nucleaseactivity, site-specific integration into a genome, helicase activity,disease symptom, binding to a molecule, specificity of binding of twomolecules, affinity of binding of two molecules, specificity to disease,sensitivity to disease, affinity of binding, enzyme activity, etc.) in afirst sample (or in a first subject) relative to a second sample (orrelative to a second subject), mean that the quantity of molecule, celland/or phenomenon in the first sample (or in the first subject) is notdifferent from the quantity in the second sample (or in the secondsubject) using any art-accepted statistical method of analysis. In oneembodiment, the quantity of molecule, cell and/or phenomenon in thefirst sample (or in the first subject) is from 90% to 100% (e.g., 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99/a, and 100%) of the quantityin the second sample (or in the second subject).

Reference herein to any numerical range expressly includes eachnumerical value (including fractional numbers and whole numbers)encompassed by that range. To illustrate, and without limitation,reference herein to a range of “at least 50” includes whole numbers of50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and fractional numbers50.1, 50.2 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, etc. In a furtherillustration, reference herein to a range of “less than 50” includeswhole numbers 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, etc., andfractional numbers 49.9, 49.8, 49.7, 49.6, 49.5, 49.4, 49.3, 49.2, 49.1,49.0, etc. In yet another illustration, reference herein to a range offrom “5 to 10” includes each whole number of 5, 6, 7, 8, 9, and 10, andeach fractional number such as 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, etc.

BRIEF SUMMARY OF THE INVENTION

The AAV Rep78 protein is required for SSI although it displays aninhibitory effect on virus replication in hybrid viruses (e.g., Ad/AAVviruses). To date, prior art strategies to construct hybrid viruses(e.g., Ad/AAV) by controlling Rep expression have met with limitedsuccess. The invention provides the discovery that AAV Rep's cis-actinginhibitory effect on hybrid virus replication and/or replicationcompetence and/or infectivity and/or productive infectivity is due to arole of an inhibitory sequence within the Rep ORF.

The inventors discovered dramatic results when comparing the expressionof Scrambled Rep78 and/or Deoptimized Rep78 with wild-type Rep78 ORFswithin a first generation Adenovirus backbone (Ad/Scr, Ad/Deopt andAd/wtRep). In particular, where Ad/wtRep was incapable of replication,Ad/Scr and Ad/Deopt replicated at comparable levels to other firstgeneration Ad, thus demonstrating a clear role for a sequence specificsignal within the wild-type Rep78 ORF in the inhibition of virusreplication. Modification of this signal (e.g., scrambled and/ordeoptimized sequences) allowed virus replication and tolerance of a highlevel of Rep protein expression. The inventors localized the inhibitorysignal to an approximately 135 bp sequence within the Rep ORF. Theidentification of a sequence specific inhibitory signal for AAV Repmediated inhibition of Ad replication explains the prior art'sinconsistent and often frustrating results obtained with production ofhybrid viruses (e.g., Ad/AAV) over the years and paves the way for theproduction of rAAV and hybrid viruses (e.g., Ad/AAV), such as in genetherapy and vaccine applications.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides adeno-associated virus (AAV) replication (Rep)sequences. In one embodiment, the invention provides nucleotidesequences encoding a chimeric protein, wherein the encoded chimericprotein contains a wild type AAV Rep inhibitory amino acid sequence, andwherein the nucleotide sequences contain a scrambled and/or deoptimizedpolynucleotide sequence encoding the wild type AAV Rep inhibitory aminoacid sequence. The invention provides vectors, cells, and virusescontaining the invention's sequences. Also provided are methods fordetecting portions of the AAV Rep inhibitory amino acid sequence, whichreduce replication and/or infection and/or productive infection byviruses. The invention's compositions and methods are useful forsite-specific integration and/or expression of heterologous sequences byrecombinant adeno-associated virus (rAAV) vectors and by rAAV virusparticles, such as hybrid viruses (e.g., Ad-AAV) comprising suchvectors. The invention's compositions and methods find application in,for example, gene therapy and/or vaccines.

The invention's methods and compositions provide a strategy for safe,site-specific gene integration that is of considerable advantage forgene therapy approaches. In one embodiment, the inventors use the uniqueability of Adeno-associated virus (AAV) genetic elements in conjunctionwith adenovirus to generate novel hybrid viruses (Ad/AAV) that retainthe capacity for stable transgene integration into a safe region of thegenome. This approach requires AAV genetic elements in conjunction withAAV Rep78, to provide the key elements for safe gene integration intothe human genome. Historically, the ability to assemble suchsite-specific integrating viruses has been limited because of the knowncellular toxicity of AAV Rep78, coupled with Rep78's inhibitory role inadenovirus replication. This has lead to difficulty in the prior art ingenerating an integrating transgene within the back-bone of a singleAd/AAV hybrid virus.

The invention also provides the discovery of an AAV nucleotide sequence(and portions thereof) in the Rep78 open reading frame (ORF) that playsa role in AAV Rep mediated inhibition of virus (e.g. Adenovirus)replication.

The invention's AAV Rep inhibitory nucleotide sequence was localized bythe inventors to a 564-bp nucleotide sequence (SEQ ID NO:17). The 564-bpnucleotide sequence includes an AAV Rep inhibitory nucleotide sequenceportion 135-bp nucleotide sequence (SEQ ID NO:01) from bp 1782 to bp1916 of the AAV2 genome, which encompasses the Rep68/40 donor splicesite and the P40 promoter region.

The inventors' discovery of the invention's AAV Rep inhibitorynucleotide sequence that mediates inhibitions of viral (e.g.,Adenoviral) replication explains the prior art's inconsistent and oftenfrustrating results reported over the years with respect to theproduction of hybrid Adenovirus/Adeno-associated virus (Ad/AAV) carryingRep.

Data herein demonstrate that, in one embodiment, the modification (e.g.,by scrambling and/or deoptimization) of the invention's AAV Repinhibitory nucleotide sequence results in removal of AAV Rep mediatedinhibition of Adenovirus replication, enabling Adenoviral replication ata level that is substantially the same as first generation Adenovirusesnot carrying Rep. Data herein also demonstrate the modification of theinvention's AAV Rep inhibitory nucleotide sequence allows Adenovirus toreplicate even in the absence of regulated Rep expression, revealing atolerance for high Rep protein expression levels.

The inventor's discovery that modifying (e.g., by scrambling and/ordeoptimization) the invention's AAV Rep inhibitory nucleotide sequence(and/or portions thereof) resulted in removal of AAV Rep mediatedinhibition of Adenovirus replication, results in the production of hightiter, stable virus (e.g., Adenovirus) carrying Rep, and paves the wayfor large scale production of hybrid AAV viruses (e.g. Ad/AAV) as wellas of recombinant AAV (rAAV) for gene therapy and vaccines.

The inventors also found that even tightly regulated Rep78 expressioncassettes that were capable of being carried by helper dependent viruseswere not viable within a ΔE1ΔE3 Ad backbone. Further, this absence ofreplication even in the presence of controlled levels of Rep contrastswith robust Adenoviral replication seen in the presence of high levelsof Rep expression produced by Rep expressing cell lines such as C12. Theinventors hypothesized that increased inhibition of replication when Repwas carried on the Adenoviral moiety could either be due to increasedexpression accompanying an increase in copy number, or due to an actualrole for the sequence of the Rep ORF.

To distinguish between these two possibilities, the inventors applied acomputer algorithm to modify the 1865 bp Rep78 DNA ORF using synonymouscodons, generating a Scrambled (Scr) and a Deoptimized (Deopt) sequence.These modified sequences are only 70-80% similar to wild-type Rep78nucleotide sequence, but encode exactly the same amino acid sequence.Further, the Deoptimized sequence specifically uses codons inunderrepresented pairs, expressing Rep78 protein at reduced levels dueto codon pair bias. Codon pair bias refers to the preference for somecodon pairs over other synonymous codons to encode the same pair ofadjacent amino acids. Utilization of underrepresented codon pairsresults in an ORF that is expressed at reduced levels, due toinefficient translation. The inventors hypothesized that modification ofthe Rep78 DNA sequence without changing the protein expressed will helpidentify any role the ORF plays in the inhibition of Adenoviralreplication. Further, comparing the ability of Adenoviruses carryingsequence modified Rep genes which express different levels of Rep, toreplicate, will help tease out the extent of the contribution of thesequence of the Rep ORF versus Rep78 protein levels on the inhibition ofAdenoviral replication.

For further clarity, the invention is further described under (A)Adeno-associated Virus and genome structure. (B) AAV Rep nucleotidesequences, (C) Vectors, (D) Viruses, (E) Cells, (F) Vaccines, (G)Exemplary applications of the invention's compositions foridentification of functional portions of wild type AAV Rep inhibitorynucleotide sequences, (H) Exemplary applications of the invention'scompositions for generation of viruses, and (1) Exemplary applicationsof the invention's compositions for expression of nucleotide sequences(e.g., in gene therapy and/or vaccine applications).

A. Adeno-Associated Virus and Genome Structure

The invention provides the discovery of useful AAV Rep nucleotidesequences. “AAV” and “Adeno-associated virus” refers to a small ssDNAvirus belonging to the family Parvoviridae, which was found to bedependent on other viruses (e.g., adenovirus, Herpes-simplex, EpsteinBarr, and cytomegalovirus) for productive infection.

AAV is exemplified by AAV2, the most widely studied AAV family member.No pathology has been convincingly linked with AAV2 infection of humans.AAV can undergo a lytic or lysogenic life cycle. Uniquely, in cellculture in the absence of helper virus functions, the virus establishesa persistent infection by integrating site-specifically into the AAVS1site on chromosome 19 (q13.3qter) of the host genome in humans andnon-human primates (135-138). However, viral sequences are also found asconcatemers of the AAV genome in an extra-chromosomal form.

The AAV2 genome (FIG. 9) is a linear single stranded DNA of about 4.7Kb. Both sense and antisense strands are packaged into virions withequal frequency. The genome contains T shaped “inverted terminalrepeats” flanking two open reading frames (ORFs), “Rep” and “Cap.” TheRep ORF encodes Rep78 (exemplified by SEQ ID NO:04 of FIG. 6 A) and thealternately spliced Rep68 (SEQ ID NO:05 of FIG. 6 B) from promoter p5and Rep52 and Rep40 from promoter 19. The p19 promoter lies within thecoding sequence for the larger Rep proteins. The Cap ORF encodes threestructural proteins VP1, VP2 and VP3 from the p40 promoter.

The genome of the AAVs has been cloned, sequenced and characterized. Forexample, the genomic sequences of AAV2 are provided in GenBank accessionNo. J01901, AF043303, and NC_001401. In general, the AAV genomecomprises about 4,700 bases and contains, at each end, an invertedrepeat region (ITR) of approximately 145 bases, serving as the origin ofreplication of the virus. The remainder of the genome is divided into 2essential regions: the left-hand part of the genome, containing the repgene involved in replication of the virus and expression of the viralgenes and; the right-hand part of the genome, containing the cap geneencoding the capsid proteins of the virus (Hearing et al., U.S. Pat. No.7,563,617).

Productive infection by AAV requires co-infection by a helper virus suchas Adenovirus. In the absence of helper virus infection, the AAVestablishes a latent infection by site-specifically integrating into theAAVS1 site on the q arm of the 19^(th) chromosome (19q13.3qter).

In particular, the invention's AAV nucleotide and amino acid sequences,as well as vectors, viruses (e.g., rAAV particles, and hybrid AAVparticles such as Ad/AAV), and cells containing one or more of thesesequences, are characterized by, among other things, containing anadeno-associated virus terminal repeat sequence. The terms “terminalrepeat,” “TR,” “intact TR,” and “full-length TR” when in reference to anadeno-associated virus (AAV) sequence, are used interchangeably to referto a nucleotide sequence which is located at each end of the AAVsingle-stranded DNA genome, and which is the only cis-acting elementrequired for genome replication and packaging. The AAV TR, in thepresence of either Rep68 or Rep78, is sufficient for site-specific viralDNA integration. Alternatively, the AAV TR refers to a nucleotidesequence which is derived from an AAV and which is involved in AAV DNAreplication, AAV DNA excision, or AAV DNA packaging into virus. In apreferred embodiment, the AAV TR is derived from AAV2 strain and isexemplified by the 145-bp sequence [5′-ttggccactc cctctctgeg cgetcgctcgetcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctcagtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttcct-3′ (SEQ IDNO:10)] from nucleotide 1 to nucleotide 145 of the AAV2 genomic sequenceof GenBank No. J01901 (Hearing et al., U.S. Pat. No. 7,563,617). The AAVTR exist in nature as an “inverted terminal repeat” (“ITR”) in a flip orflop orientation, based on the orientation of the internal palindromewith respect to the D sequence. The AAV ITR contains a hairpin structurethat appears to contain hot spots of integration.

“Rep” and “replication protein” when in reference to an adeno-associatedvirus (AAV) protein sequence, refers to a protein encoded by a AAV repgene region, and is exemplified by AAV Rep proteins Rep78, Rep68, Rep52and Rep40.

The “rep gene region” of the adeno-associated virus genome refers to anucleotide sequence that is derived from an adeno-associated virus, andwhich encodes one or more Rep proteins. AAV Rep proteins include Rep78,Rep68, Rep52 and Rep40.

“Rep68” and “Rep78,” are Rep sequences produced from unspliced andspliced transcripts from the p5 promoter. In one embodiment, Rep78 isexemplified by the wild type Rep78 SEQ ID NO:04 (FIG. 6, A) encoded bythe DNA sequence SEQ ID NO:03 (FIG. 5). In another embodiment, Rep68 isexemplified by wild type Rep68 SEQ ID NO:05 (FIG. 6, B). Rep78 and Rep68are multifunctional proteins with largely overlapping functions inalmost every stage of the AAV life cycle, such as site-specific DNAbinding, helicase activity, and/or site-specific endonuclease activity.Rep78 and/or Rep68 are required in trans for AAV replication and/orexcision from the host genome (U.S. Pat. No. 5,658,776; Trempe et al.,U.S. Pat. No. 5,837,484; Burstein et al., WO 98/27207; Johnson et al.,U.S. Pat. No. 5,658,785; Carter, U.S. Pat. No. 7,785,888). Thus, a“functional” Rep protein (and/or portion thereof) refers to a protein(and/or portion thereof) that has one or more Rep activity exemplifiedby site-specific DNA binding, helicase activity, and site-specificendonuclease activity.

“AAV Rep inhibitory amino acid sequence” refers to the amino acidsequence encoded by the 135-bp DNA sequence from bp 1782 to bp 1916 ofthe AAV2 genome (FIG. 4 B).

“Rep-mediated excision” and “Rep-mediated nuclease” when in referencethe activity by a Rep protein, or functional fragment thereof,interchangeably refer to the endonuclease activity of the protein inproducing two or more fragments of a substrate nucleotide sequence.Methods for determining Rep-mediated excision activity are known in theart, and described herein. For example, data herein demonstrate thatadenovirus Ad/sRep78 (containing a scrambled Rep78 sequence) andadenovirus Ad/dRep78 (containing a deoptimized Rep78 sequence) retainedRep-mediated excision activity as demonstrated by cleavage of a foldedAAV ITR as substrate (Example 3).

“Rep52” and “Rep40” are Rep sequences produced from unspliced andspliced transcripts respectively, from the p19 promoter. All four Repproteins possess helicase and ATPase activity. In addition, Rep68 andRep78 are capable of site-specific binding to the ribosome binding site(RBS) and have site-specific endonuclease activity, required forseparation of replicated viral genomes.

The “Cap gene” of adeno-associated virus refers to a gene encoding VP1,VP2 and VP3 structural proteins making up the AAV capsid. The exemplaryAAV-2 “AAV capsid” comprises 60 viral capsid proteins arranged into anicosahedral structure, with VP1, VP2 and VP3 present in approximately a1:1:8 molar ratio.

B. AAV Rep Nucleotide Sequences

The inventors have discovered that first generation adenovirusescarrying AAV Rep78 expressed under a tetracycline inducible system wereincapable of growing (Example 2). This contradicts prior art reports ofthe production of a helper dependent Ad carrying Rep78 (Recchia et al.(2004) Molecular Therapy 10(No. 4):660-670). A similar lack of thereproducibility of generating replicative virus carrying AAV Rep78 isdemonstrated by the prior art's failure to construct a first generationAd carrying Rep expressed under the al antitrypsin promoter by one group(Carlson et al. (2002) Molecular Therapy 6(1):91-98), in contrast toreports that a helper dependent Ad carrying Rep78 expressed under an alantitrypsin promoter was capable of growing (Recchia et al. (1999) PNAS96:2615-2620).

Thus, in one embodiment, the invention provides AAV recombinantnucleotide sequences (as well as vectors, viruses (e.g., rAAV, andhybrid AAV), and cells containing one or more these sequences) encodinga chimeric protein, a) wherein the encoded chimeric protein i) comprisesat least a portion of wild type AAV Rep inhibitory amino acid sequencelisted as SEQ ID NO:20 (i.e., the 564-bp DNA sequence from bp1623 to bp2186 of the AAV2 genome) and/or SEQ ID NO:02 (i.e., the 135-bp DNAsequence from bp 1782 to bp 1916 of the AAV2 genome, FIG. 4 B), and ii)has Rep-mediated nuclease activity, and b) wherein the recombinantnucleotide sequence comprises a scrambled polynucleotide sequenceencoding wild type AAV Rep inhibitory amino acid sequence listed as SEQID NO:20 and/or SEQ ID NO:02 (and/or portion thereof).

One advantage of the invention's AAV nucleotide and amino acidsequences, as well as vectors, viruses (e.g., rAAV particles, and hybridAAV particles such as Ad/AAV), and cells containing one or more of thesesequences, is that they are useful in gene therapy (including genetransfer for monogenic disorders such as hemophilia, and polygenicdisease such as cancer) and vaccines. The invention's AAV nucleotide andamino acid sequences, as well as vectors, viruses (e.g., rAAV particles,and hybrid AAV particles such as Ad/AAV), and cells containing one ormore of these sequences, can be used to express various heterologousgene products in host cells by transformation and transduction,respectively.

Another advantage of the invention's AAV nucleotide and amino acidsequences, as well as vectors, viruses (e.g., rAAV particles, and hybridAAV particles such as Ad/AAV), and cells containing one or more of thesesequences, is that they retain Rep-mediated nuclease activity, yet lackthe AAV Rep inhibitory nucleotide sequence that inhibits viralproductive replication.

Yet a further advantage of the invention's AAV nucleotide and amino acidsequences, as well as vectors, viruses (e.g., rAAV particles, and hybridAAV particles such as Ad/AAV), and cells containing one or more of thesesequences, is that they provide hybrid viruses (e.g. Ad-AAV hybridvirus) carrying the AAV ITR flanked transgene and/or the AAV Rep-Capcoding sequences, which allows efficient co-infection of 293 cells,eliminate the need for producer cell lines and would greatly ease theproduction of high titer AAV. These hybrid viruses also retain thecapacity for stable transgene integration into a safe region of thegenome.

Another advantage of the invention's AAV nucleotide and amino acidsequences, as well as vectors, viruses (e.g., rAAV particles, and hybridAAV particles such as Ad/AAV), and cells containing one or more of thesesequences, is that they provide hybrid viruses (e.g. Ad-AAV hybridvirus) that can tolerate Rep78 (or Rep68) within the viral background,which provides a unique opportunity to place all genetic elements in asingle virus for the purpose of safely integrating a transgene into asafe region of the human genome. This is a significant advance in thefield as it provides a safer alternative to retroviruses andlentiviruses in gene replacement strategies.

Data herein demonstrate that the inhibitory function of the wild typeAAV Rep inhibitory nucleotide sequence, exemplified by SEQ ID NO:01, onvirus replication was abolished when using either a) a scrambled AAV Repinhibitory nucleotide sequence that did NOT alter the level of expressedRep78 or Rep68, or b) a deoptimized AAV Rep inhibitory nucleotidesequence that DID reduce the level of expressed Rep78 and/or Rep68. Inparticular, data herein demonstrate that the exemplary adenovirusAd/sRep78 (containing a scrambled Rep78 sequence) and adenovirusAd/dRep78 (containing a deoptimized Rep78 sequence) retainedRep-mediated nuclease activity as demonstrated by cleavage of a foldedAAV ITR as substrate (Example 3).

The invention's AAV nucleotide and amino acid sequences, as well asvectors, viruses (e.g., rAAV particles, and hybrid AAV particles such asAd/AAV), and cells containing one or more of these sequences, carryingAAV elements on the backbone of a larger virus are useful not only forthe production of rAAV, but also as potential integrating gene transfervectors. The only difference in Rep expression in such vectors would bethe requirement for both Rep68/78 and Rep52 expression for rAAVproduction, versus only Rep68/78 expression needed for integration.

Production of a rAAV possessing AAV's unique ability of Rep mediatedsite-specific integration is not feasible, due to the small size of itsgenome and the toxic effects of Rep protein on the host cell (Winocouret al. (1988) Perturbation of the cell cycle by adeno-associated virus.Virology 167:393-399). Introduction of a Rep cassette into the rAAVwould further reduce the viable transgene size to about 3 Kb. Further,since the entire cassette flanked by the AAV ITR integrates in thepresence of Rep protein, an internal Rep cassette would also beintegrated. Alternatively, since the only elements required forsite-specific integration have been shown to be the AAV ITR or IEE incis and the Rep68/78 protein in trans, an AAV ITR flanked transgenecassette and a Rep expression cassette outside the AAV TR's could becarried on the backbone of a larger virus (such as adenovirus, herpessimplex virus, retrovirus, lentivirus, baculovirus, etc.), combiningAAV's ability to site-specifically integrate with the large transgenesize of a larger virus.

In one embodiment, the invention's AAV nucleotide and amino acidsequences, as well as vectors, viruses (e.g., rAAV particles, and hybridAAV particles such as Ad/AAV), and cells containing one or more of thesesequences, are useful in gene therapy (including gene transfer formonogenic disorders such as hemophilia, and polygenic disease such ascancer) and vaccines.

i. Heterologous Polynucleotide Sequences

The invention's compositions can be used to express various heterologousgene products in host cells by transduction. Thus, in one embodiment,the recombinant nucleotide sequence comprises a heterologouspolynucleotide sequence operably linked to a first AAV ITR. In a morepreferred embodiment, the heterologous polynucleotide sequence isflanked by the first AAV ITR and by a second AAV ITR.

The terms “flanking,” and “flank” when made in reference to a first andsecond nucleotide sequences (e.g., inverted terminal repeats (ITRs)) inrelation to a third nucleotide sequence (e.g., a DNA sequence ofinterest) mean that the first nucleotide sequence is linked to the 5′end (i.e., upstream) of the third sequence, and the second nucleotidesequence is linked to the 3′ end (i.e., downstream) of the thirdsequence, preferably (although not necessarily) without any interveningsequences of viral origin in order to reduce the likelihood ofrecombination. Recent evidence suggests that a single ITR can besufficient to carry out the functions normally associated withconfigurations comprising two ITRs (U.S. Pat. No. 5,478,745), and vectorconstructs with only one ITR can thus be employed in conjunction withthe packaging and production methods described herein. The resultantrecombinant viral vector is referred to as being “defective” in viralfunctions when specific viral coding sequences are deleted from thevector (Carter, U.S. Pat. No. 7,785,888).

In preferred embodiments, the invention's sequences have Rep-mediatednuclease activity.

In a particular embodiment, the recombinant nucleotide sequence furthercomprises a nucleic acid sequence encoding one or more AAV capsidproteins.

ii. Scrambled AAV Rep Inhibitory Nucleotide Sequences, IncludingDeoptimized AAV Rep Inhibitory Nucleotide Sequences

In preferred embodiments, the invention's AAV nucleotide and amino acidsequences, as well as vectors, viruses (e.g., rAAV particles, and hybridAAV particles such as Ad/AAV), and cells containing one or more of thesesequences, comprise a scrambled polynucleotide sequence encoding atleast a portion of the wild type AAV Rep inhibitory amino acid sequencelisted as SEQ ID NO:20 and/or SEQ ID NO:02.

Without intending to limit the particular sequence of the scrambledpolynucleotide sequence encoding at least a portion of the wild type AAVRep inhibitory amino acid sequence, in a particular embodiment, thescrambled polynucleotide sequence comprises SEQ ID NO:18 (FIG. 4G)and/or SEQ ID NO:07 (FIG. 4 C and FIG. 7 A).

In another embodiment, the scrambled polynucleotide sequence comprises adeoptimized AAV Rep inhibitory nucleotide sequence. While not limitingthe exact sequence of the deoptimized AAV Rep inhibitory nucleotidesequence, in some embodiments, the deoptimized AAV Rep inhibitorynucleotide sequence comprises SEQ ID NO:19 (FIG. 4H) and/or SEQ ID NO:09(FIG. 4 D, FIG. 7 B) and/or portion thereof.

In particular embodiment, the scrambled polynucleotide sequence thatencodes at least a portion of the wild type AAV Rep inhibitory aminoacid sequence, has from 66% to 99% identity to the wild type AAV Repinhibitory nucleotide sequence listed as SEQ ID NO:17 (FIG. 4E, FIG.12A-C, FIG. 13A-C) and/or SEQ ID NO:01 (FIG. 4 A, FIG. 12A-C, FIG.13A-C). This includes identities of 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%. Ina particular embodiment, the scrambled polynucleotide sequence thatencodes at least a portion of the wild type AAV Rep inhibitory aminoacid sequence, has from 70% to 80% identity to the wild type AAV Repinhibitory nucleotide sequence listed as SEQ ID NO:17 and/or SEQ IDNO:01.

In a particular embodiment, the invention's AAV nucleotide and aminoacid sequences, as well as vectors, viruses (e.g., rAAV particles, andhybrid AAV particles such as Ad/AAV), and cells containing one or moreof these sequences, are isolated. The terms “purified,” “isolated,” andgrammatical equivalents thereof refer to the reduction in the amount ofat least one undesirable component (such as cell, protein, nucleic acidsequence, carbohydrate, etc.) from a sample, including a reduction byany numerical percentage of from 5% to 100%, such as, but not limitedto, from 10% to 100%, from 20% to 100%, from 30% to 100%, from 40% to100%, from 50% to 100%, from 60% to 100%, from 70% to 100%, from 80% to100%, and from 90% to 100%. Thus purification results in “enrichment,”i.e., an increase in the amount of a desirable component cell, protein,nucleic acid sequence, carbohydrate, etc.) relative to the undesirablecomponent. For example, “purifying” encapsidated vectors refers to theisolation of the encapsidated vectors in a more concentrated form(relative to the starting material, such as the cell lysate and/orextracellular solution), e.g., using CsCl₂ density gradients.

iii. Therapeutic Sequences for Gene Therapy and Vaccines

In particular embodiments, the heterologous polynucleotide sequencecontained in the invention's AAV nucleotide and amino acid sequences, aswell as vectors, viruses (e.g., rAAV particles, and hybrid AAV particlessuch as Ad/AAV), and cells containing one or more of these sequences,comprises a therapeutic nucleotide sequence. A “therapeutic nucleotidesequence” is a DNA sequence and/or RNA sequence of therapeutic interest,including sequences that encode a protein of therapeutic interest.

Therapeutic nucleotide sequences are exemplified by, but not limited to,sequences encoding a disease associated polypeptide, and/or encoding anantigen polypeptide.

Thus, in one embodiment, the therapeutic nucleotide sequences containedin the invention's AAV nucleotide and amino acid sequences, as well asvectors, viruses (e.g., rAAV particles, and hybrid AAV particles such asAd/AAV), and cells containing one or more of these sequences, comprisesa therapeutic sequence that encodes a “disease associated” polypeptide,meaning a polypeptide whose level (e.g., presence, absence, increase,and/or decrease relative to a control, etc.) that is correlated withdisease and/or with risk of disease based on family history, geneticfactors, environmental factors, etc.

Illustrative therapeutic nucleotide sequences that encode diseaseassociated polypeptides include, but are not limited to, sequences whichencode enzymes; lymphocytes (e.g., interleukins, interferons, TNF,etc.); growth factors (e.g., erythropoietin, G-CSF, M-CSF, GM-CSF,etc.); neurotransmitters or their precursors or enzymes responsible forsynthesizing them; trophic factors (e.g., BDNF, CNTF, NGF, IGF, GMF,aFGF, bFGF, NT3, NT5, HARP/pleiotrophin, etc.); apolipoproteins (e.g.,ApoAI, ApoAIV, ApoF, etc.); lipoprotein lipase (LPL); thetumor-suppressing genes (e.g., p53, Rb, Rap1A, DCC k-rev, etc.); factorsinvolved in blood coagulation (e.g., Factor VII, Factor VIII, Factor IX,etc.); DNA repair enzymes; suicide genes (thymidine kinase or cytosinedeaminase); blood products; hormones; etc. (Hearing et al. U.S. Pat. No.7,563,617).

In one preferred embodiment, the therapeutic disease associatednucleotide sequence encodes a wild type gene for which a mutant has beenassociated with a human disease. Such wild type genes are exemplified,but not limited to, the adenosine deaminase (ADA) gene (GenBankAccession No. M13792) associated with adenosine deaminase deficiencywith severe combined immune deficiency; alpha-1-antitrypsin gene(GenBank Accession No. M11465) associated with alpha1-antitrypsindeficiency; beta chain of hemoglobin gene (GenBank Accession No.NM_000518) associated with beta thalassemia and Sickle cell disease;receptor for low density lipoprotein gene (GenBank Accession No. D16494)associated with familial hypercholesterolemia; lysosomalglucocerebrosidase gene (GenBank Accession No. K02920) associated withGaucher disease; hypoxanthine-guanine phosphoribosyltransferase (HPRT)gene (GenBank Accession No. M26434, J00205, M27558, M27559, M27560,M27561, M29753, M29754, M29755, M29756, M29757) associated withLesch-Nyhan syndrome; lysosomal arylsulfatase A (ARSA) gene (GenBankAccession No. NM_000487) associated with metachromatic leukodystrophy;ornithine transcarbamylase (OTC) gene (GenBank Accession No. NM_000531)associated with ornithine transcarbamylase deficiency; phenylalaninehydroxylase (PAH) gene (GenBank Accession No. NM_000277) associated withphenylketonuria; purine nucleoside phosphorylase (NP) gene (GenBankAccession No. NM_000270) associated with purine nucleoside phosphorylasedeficiency; the dystrophin gene (GenBank Accession Nos. M18533, M17154,and M18026) associated with muscular dystrophy; the utrophin (alsocalled the dystrophin related protein) gene (GenBank Accession No.NM_007124) whose protein product has been reported to be capable offunctionally substituting for the dystrophin gene; and the human cysticfibrosis transmembrane conductance regulator (CFTR) gene (GenBankAccession No. M28668) associated with cystic fibrosis. In a particularembodiment, the disease associated polypeptide of interest is a cancerderived antigen such as carcino-embryonic antigen (CEA) and her2neuantigen. In a preferred embodiment, the therapeutic gene is human FactorVIII (Hearing et al. U.S. Pat. No. 7,563,617).

In a particular embodiment, the therapeutic disease associatednucleotide sequence encodes an antisense RNA or a ribozyme (Carter, U.S.Pat. No. 7,785,888). In yet another embodiment, the therapeutic diseaseassociated nucleotide sequence is selected from the group of (i) apolynucleotide encoding a protein useful in gene therapy to relievedeficiencies caused by missing, defective or sub-optimal levels of astructural protein or enzyme; (ii) a polynucleotide that is transcribedinto an anti-sense molecule; (iii) a polynucleotide that is transcribedinto a decoy that binds a transcription or translation factor, (iv) apolynucleotide that encodes a cellular modulator; (v) a polynucleotidethat can make a recipient cell susceptible to a specific drug; (vi) apolynucleotide for cancer therapy; and (vii) a polynucleotide thatencodes an antigen or antibody. (Carter, U.S. Pat. No. 7,785,888).

In some applications, the therapeutic disease associated nucleotidesequence is selected from the group of herpes virus thymidine kinasegene, E1A tumor suppressor gene, and p53 tumor suppressor gene (Carter,U.S. Pat. No. 7,785,888). In other applications, the therapeutic diseaseassociated nucleotide sequence encodes a protein selected from the groupof cytosine deaminase (CD), herpes simplex-virus thymidine kinase(HSV-TK), DNA-binding domain (DBD) of poly(ADP-ribose) polymerase(PARP), cytotoxic protease 2A and 3C (Ktpper et al., U.S. Pat. No.7,351,697), Factor VITA, Factor VIII and Factor IX (Scaria, U.S. Pat.No. 7,307,068).

In some embodiments, the therapeutic disease associated nucleotidesequence encodes a protein selected from the group of Cystic FibrosisTransmembrane Conductance Regulator (CFTR), coagulation factor FIX,hRPE65v2, neurotrophic factor Neurturin (NTN), and al antitrypsin. TheFDA has approved clinical trials for gene therapy using earliergeneration rAAV viruses expressing these proteins. In a particularembodiment, the therapeutic disease associated nucleotide sequenceencodes a human Factor VIII (FVIII) gene (Example 1).

In one embodiment, the therapeutic nucleotide sequences contained in theinvention's AAV nucleotide and amino acid sequences, as well as vectors,viruses (e.g., rAAV particles, and hybrid AAV particles such as Ad/AAV),and cells containing one or more of these sequences, comprises atherapeutic sequence that encodes an antigen polypeptide.

The terms “antigen,” “immunogen,” “antigenic,” “immunogenic,”“antigenically active,” “immunologic,” and “immunologically active” whenmade in reference to a molecule, refer to any substance that is capableof inducing a specific humoral immune response (including eliciting asoluble antibody response) and/or cell-mediated immune response(including eliciting a CTL response). In one embodiment the antigen isexemplified by Human Immunodeficiency virus gag protein, malariacircumsporozite protein (CSP_(full)) antigen, malaria CSP T cell epitope(SEQ ID NO:12; EYLNKIQNSLSTEWSPCSVT; U.S. Pat. No. 6,669,945), malariaCSP B Cell epitope (SEQ ID NO:13; NANPNANPNANPNANPNANPNANP; WO2009/082440 A2), and Pseudomonas antigen.

In some embodiments, the antigen polypeptide is “pathogen derived,”meaning expressed by a pathogen (e.g., bacteria, virus, parasite,protozoan, fungus, etc.), such as Herpes virus, Neisseria gonorrhea,Treponema, Escherichia coli, Respiratory Syncytial virus, tuberculosis,Streptococcus, Chlamydia, and Ebola virus. Pathogen derived antigens areexemplified by Human Immunodeficiency virus (HIV) gag protein (includingthe HXB2 strain gag protein (Genbank Accession #K03455), HIV Gag proteinantigen such as HIV Gap protein immunodominant peptide AMQMLKETI (SEQ IDNO:14; WO 2010/051820 A1), HIV Pol protein antigen, HIV Nef proteinantigen, malaria circumsporozite protein (CSP_(full)) antigen, malariaCSP T cell epitope (SEQ ID NO:12; EYLNKIQNSLSTEWSPCSVT; U.S. Pat. No.6,669,945), malaria CSP B Cell epitope (SEQ ID NO:13;NANPNANPNANPNANPNANPNANP; WO 2009/082440 A2), and Pseudomonas antigen.

In a particular embodiment, the antigen comprises an epitope. The terms“epitope” and “antigenic determinant” refer to a structure on anantigen, which interacts with the binding site of an antibody or T cellreceptor as a result of molecular complementarity. An epitope maycompete with the intact antigen, from which it is derived, for bindingto an antibody. Generally, secreted antibodies and their correspondingmembrane-bound forms are capable of recognizing a wide variety ofsubstances as antigens, whereas T cell receptors are capable ofrecognizing only fragments of proteins which are complexed with MHCmolecules on cell surfaces. Antigens recognized by immunoglobulinreceptors on B cells are subdivided into three categories: T-celldependent antigens, type 1 T cell-independent antigens; and type 2 Tcell-independent antigens. Also, for example, when a protein or fragmentof a protein is used to immunize a host animal, numerous regions of theprotein may induce the production of antibodies which bind specificallyto a given region or three-dimensional structure on the protein; theseregions or structures are referred to as antigenic determinants. Anantigenic determinant may compete with the intact antigen (i.e., theimmunogen used to elicit the immune response) for binding to anantibody.

Exemplary epitopes include, without limitation YPYDVPDYA (SEQ ID NO:15;U.S. Pat. No. 7,255,859), EphrinA2 epitopes from renal cell carcinomaand prostate cancer (U.S. Pat. No. 7,297,337), hepatitis C virusepitopes (U.S. Pat. Nos. 7,238,356 and 7,220,420), vaccinia virusepitopes (U.S. Pat. No. 7,217,526), dog dander epitopes (U.S. Pat. No.7,166,291), human papilloma virus (HPV) epitopes (U.S. Pat. Nos.7,153,659 and 6,900,035), Mycobacterium tuberculosis epitopes (U.S. Pat.Nos. 7,037,510 and 6,991,797), bacterial meningitis epitopes (U.S. Pat.No. 7,018,637), malaria epitopes (U.S. Pat. No. 6,942,866), and type 1diabetes mellitus epitopes (U.S. Pat. No. 6,930,181).

C. Vectors

The invention provides vectors comprising one or more of the invention'srecombinant nucleotide sequences. “Vector” and “vehicle” refer to anagent that contains and/or transfers genetic material to a cell,including for example linear DNA, encapsidated virus particles,liposomes, bacteriophages, plasmids, and any combination thereof. Inother embodiments, the recombinant viral vector sequence is provided inthe host cells transfected with the viral vector. A vector may be usedto transfer, introduce and/or insert exogenous modified genetic material(as recombinant DNA) into the genome of a recipient (host) cell.Delivery of genetic material by a “viral vector” is termed“transduction,” with the infected cells described as “transduced.” Thisprocess can be performed inside a living organism (in vivo) or in cellculture (in vitro and/or ex vivo). Viral based gene transfer/vectorsinclude, for example, adenovirus, adeno-associated virus, retroviruses,alpha viruses, lentiviruses, vaccinia viruses, baculoviruses, fowl poxand herpes viruses.

Vectors (such as linear DNA, encapsidated virus particles, liposomes,bacteriophages, plasmids, etc.) may be introduced into cells usingtechniques well known in the art. The term “introducing” a nucleic acidsequence into a cell refers to the introduction of the nucleic acidsequence into a target cell to produce a “transformed” or “transgenic”cell. Methods of introducing nucleic acid sequences into cells are wellknown in the art. For example, where the nucleic acid sequence is aplasmid or naked piece of linear DNA, the sequence may be “transfected”into the cell. Alternatively, where the nucleic acid sequence isencapsidated into a viral particle, the sequence may be introduced intoa cell by “transduction,” i.e., the introduction of the nucleic acidsequence into the cell by “infection” with a virus containing thenucleic acid sequence, e.g., as part of a recombinant viral genome.

“Transfect” and “transfecting” refer to any mechanism by which a vectormay be incorporated into a host cell. For example, where the vector is aplasmid or naked piece of linear DNA, the vector may be transfected intothe cell using, for example, calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofection,protoplast fusion, and biolistics. In one embodiment, successfultransfection results in the capability of the host cell to express anyoperative genes carried by the vector. Transfections may be stable ortransient. One example of a transient transfection comprises vectorexpression within a cell, wherein the vector is not integrated withinthe host cell genome. Alternatively, a stable transfection comprisesvector expression within a cell, wherein the vector is integrated withinthe host cell genome.

Transformation of a cell with the invention's vectors may be stable ortransient. The terms “transient transformation” and “transientlytransformed” refer to the introduction of one or more nucleotidesequences of interest into a cell in the absence of integration of thenucleotide sequence of interest into the host cell's genome. Transienttransformation may be detected by, for example, enzyme-linkedimmunosorbent assay (ELISA) that detects the presence of a polypeptideencoded by one or more of the nucleotide sequences of interest.Alternatively, transient transformation may be detected by detecting theactivity of the protein encoded by the nucleotide sequence of interest.The term “transient transformant” refer to a cell that has transientlyincorporated one or more nucleotide sequences of interest. In contrast,the terms “stable transformation” and “stably transformed” refer to theintroduction and integration of one or more nucleotide sequence ofinterest into the genome of a cell. Thus, a “stable transformant” isdistinguished from a transient transformant in that, whereas genomic DNAfrom the stable transformant contains one or more heterologousnucleotide sequences of interest, genomic DNA from the transienttransformant does not contain the heterologous nucleotide sequence ofinterest. Stable transformation of a cell may be detected by Southernblot hybridization of genomic DNA of the cell with nucleic acidsequences that are capable of binding to one or more of the nucleotidesequences of interest. Alternatively, stable transformation of a cellmay also be detected by the polymerase chain reaction of genomic DNA ofthe cell to amplify the nucleotide sequence of interest.

“Gene expression” refers to the process of converting geneticinformation encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, orsnRNA) through “transcription” of the gene (i.e., via the enzymaticaction of an RNA polymerase), and for protein encoding genes, intoprotein through “translation” of mRNA. Gene expression can be regulatedat many stages in the process. “Up-regulation” or “activation” refers toregulation that increases the production of gene expression products(i.e., RNA or protein), while “down-regulation” or “repression” refersto regulation that decrease production. Molecules (e.g., transcriptionfactors) that are involved in up-regulation or down-regulation are oftencalled “activators” and “repressors,” respectively.

In some embodiments, the invention's recombinant nucleotide sequence iscomprised in an expression vector. Thus, in a particular embodiment, theinvention provides expression vectors comprising a) one or more of therecombinant nucleotide sequences described herein, and b) a heterologouspolynucleotide sequence operably linked to a first AAV ITR. Therecombinant nucleotide sequence of the invention's expression vectors isexemplified by, but not limited to, a sequence encoding a chimericprotein, a) wherein the encoded chimeric protein i) comprises at least aportion of wild type AAV Rep inhibitory amino acid sequence listed asSEQ ID NO:20 (i.e., encoded by the 564-bp DNA sequence from bp 1623 tobp 2186 of the AAV2 genome) and/or SEQ ID NO:02 (i.e., encoded by the135-bp DNA sequence from bp 1782 to bp 1916 of the AAV2 genome, FIG. 4B), and ii) has Rep-mediated nuclease activity, and b) wherein therecombinant nucleotide sequence comprises a scrambled polynucleotidesequence encoding the at least a portion of the wild type AAV Repinhibitory amino acid sequence listed as SEQ ID NO:20 and/or SEQ IDNO:02. In a particular embodiment the heterologous polynucleotidesequence is flanked by the first AAV ITR and by a second AAV ITR.

The term “expression vector” as used herein refers to a nucleotidesequence containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression (i.e., transcription intoRNA and/or translation into a polypeptide) of the operably linked codingsequence in a particular host cell. Expression vectors are exemplifiedby, but not limited to, plasmid, phagemid, shuttle vector, cosmid,virus, chromosome, mitochondrial DNA, plastid DNA, and nucleic acidfragments thereof. Nucleic acid sequences used for expression inprokaryotes include a promoter, optionally an operator sequence, aribosome binding site and possibly other sequences. Eukaryotic cells areknown to utilize promoters, enhancers, and termination andpolyadenylation signals. Expression vectors include “gene therapy viralvectors,” viral vectors comprising a therapeutic nucleotide sequence.

In a particular embodiment, the expression vector is a viral vector, asexemplified by, but not limited to a gene therapy viral vector.

In another particular embodiment, the invention's expression vectorscontain the scrambled polynucleotide sequence that encodes at least aportion of the wild type AAV Rep inhibitory amino acid sequence, inoperable combination with a promoter.

The term “promoter,” “promoter element,” or “promoter sequence” as usedherein, refers to a DNA sequence that, when ligated to a nucleotidesequence of interest, is capable of controlling the transcription of thenucleotide sequence of interest into mRNA. The terms “control,” “drive,”“regulate,” and “facilitate” when used in reference to a promoter areinterchangeably used to refer to the activity of the promoter inbringing about and/or altering the level of transcription of an operablylinked nucleotide sequence. A promoter is typically, though notnecessarily, located 5′ (i.e., upstream) of a nucleotide sequence ofinterest whose transcription into mRNA it controls, and provides a sitefor specific binding by RNA polymerase and other transcription factorsfor initiation of transcription.

Promoters may be inducible or constitutive. “Inducible” and“regulatable” promoter interchangeably refer to a promoter that iscapable of directing a level of transcription of an operably linkednucleic acid sequence in the presence of a stimulus (e.g., heat shock,chemicals, etc.) that is different from the level of transcription ofthe operably linked nucleic acid sequence in the absence of thestimulus. Inducible promoters are exemplified by lac, tac, trc, ara,trp, X phage, T7 phage, and T5 phage promoter, and tetracyclineinducible promoters. In a particularly preferred embodiment, theinducible promoter is a tetracycline inducible promoter (Example 2).

“Constitutive” promoter means that the promoter is capable of directingtranscription of an operably linked nucleic acid sequence in the absenceof a stimulus (e.g., heat shock, chemicals, etc.). Constitutivepromoters are exemplified by vaccinia virus p7.5 promoter. Genetranscription in viruses (such as poxvirus) is temporally regulated, andtherefore contain promoters for early, intermediate, and late geneexpression. Certain virus (e.g., poxvirus) genes are expressedconstitutively, and promoters for these “early-late” genes bear hybridstructures. Synthetic early-late promoters have also been developed. SeeHammond J. M., et al., J. Virol. Methods 66:135-8 (1997); ChakrabartiS., et al., Biotechniques 23:1094-7 (1997). Examples of early promotersinclude the 7.5-kD promoter (also a late promoter), the DNA polpromoter, the tk promoter, the RNA pol promoter, the 19-kD promoter, the22-kD promoter, the 42-kD promoter, the 37-kD promoter, the 87-kDpromoter, the H3′ promoter, the H6 promoter, the D1 promoter, the D4promoter, the D5 promoter, the D9 promoter, the D12 promoter, the 13promoter, the M1 promoter, and the N2 promoter. See, e.g., Moss, B.,“Poxviridae and their Replication” IN Virology, 2d Edition, B. N.Fields, D. M. Knipe et al., Eds., Raven Press, p. 2088 (1990). Exampleof late promoters include the 7.5-kD promoter, the MIL promoter, the37-kD promoter, the 11-kD promoter, the 11L promoter, the 12L promoter,the 13L promoter, the 15L promoter, the 17L promoter, the 28-kDpromoter, the HI L promoter, the H3L promoter, the H5L promoter, the H6Lpromoter, the H8L promoter, the D11L promoter, the D12L promoter, theD13L promoter, the A1L promoter, the A2L promoter, the A3L promoter, andthe P4b promoter. See, e.g., Moss, B., “Poxviridae and theirReplication” IN Virology, 2d Edition, B. N. Fields, D. M. Knipe et al.,Eds., Raven Press, p. 2090 (1990). Additional constitutive promotersinclude the human U1-1 small nuclear RNA promoter (pHU-1). In aparticularly preferred embodiment, the constitutive promoter is thehuman U1-1 small nuclear RNA promoter (pHU-1) (Example 4).

D. Viruses

In one particular embodiment, the invention provides recombinant viruses(e.g., rAAV, and hybrid AAV viruses) comprising one or more of theinvention's AAV recombinant nucleotide sequences. In particular, theinvention provides recombinant viruses comprising an AAV recombinantnucleotide sequence encoding a chimeric protein, a) wherein the encodedchimeric protein i) comprises at least a portion of wild type AAV Repinhibitory amino acid sequence listed as SEQ ID NO:20 (i.e., encoded bythe 564-bp DNA sequence from bp 1623 to bp 2186 of the AAV2 genome)and/or SEQ ID NO:02 (i.e., encoded by the 135-bp DNA sequence from bp1782 to bp 1916 of the AAV2 genome, FIG. 4 B), and ii) has Rep-mediatednuclease activity, and b) wherein the recombinant nucleotide sequencecomprises a scrambled polynucleotide sequence encoding the wild type AAVRep inhibitory amino acid sequence listed as SEQ ID NO:20 and/or SEQ IDNO:02 (and/or portion thereof).

Methods for production of the invention's rAAV vectors and AAVcontaining these vectors are known in the art (Carter, U.S. Pat. No.7,785,888) and described herein (Example 1).

For example, methods for achieving high titers of rAAV viruspreparations that are substantially free of contaminating virus and/orviral or cellular proteins are outlined by Atkinson et al. in WO99/11764. Techniques described therein can be employed for thelarge-scale production of rAAV viral particle preparations (Carter, U.S.Pat. No. 7,785,888).

These various examples address the production of rAAV viral particles atsufficiently high titer, minimizing recombination between rAAV vectorand sequences encoding packaging components, and producing rAAV viruspreparations that are substantially free of contaminating virus and/orviral or cellular protein (Carter, U.S. Pat. No. 7,785,888).

Optionally, rAAV virus preparations can be further processed to purify(i.e., enrich for) rAAV particles and/or otherwise render them suitablefor administration to a subject. See Atkinson et al. for exemplarytechniques (WO 99/11764). Purification techniques can include isopynicgradient centrifugation, and chromatographic techniques. Reduction ofinfectious helper virus activity can include inactivation by heattreatment or by p1H treatment as is known in the art. Other processescan include concentration, filtration, diafiltration, or mixing with asuitable buffer or pharmaceutical excipient. Preparations can be dividedinto unit dose and multi dose aliquots for distribution, which willretain the essential characteristics of the batch, such as thehomogeneity of antigenic and genetic content, and the relativeproportion of contaminating helper virus (Carter, U.S. Pat. No.7,785,888).

Various methods for the determination of the infectious titer of a viralpreparation are known in the art. For example, one method for titerdetermination is a high-throughput titering assay as provided-byAtkinson et al. (WO 99/11764). Virus titers determined by this rapid andquantitative method closely correspond to the titers determined by moreclassical techniques. In addition, however, this high-throughput methodallows for the concurrent processing and analysis of many viralreplication reactions and thus has many others uses, including forexample the screening of cell lines permissive or non-permissive forviral replication and infectivity (Carter, U.S. Pat. No. 7,785,888).

In one embodiment, the invention's recombinant viruses (e.g., rAAV, andhybrid AAV viruses) comprise a heterologous polynucleotide sequenceoperably linked to a first AAV ITR. In a particular embodiment,heterologous polynucleotide sequence is flanked by the first AAV ITR andby a second AAV ITR.

In yet another embodiment, the invention's recombinant viruses (e.g.,rAAV, and hybrid AAV viruses) further comprise a nucleic acid sequenceencoding AAV Cap

In particular embodiments, the invention's recombinant viruses (e.g.,rAAV, and hybrid AAV viruses) are characterized by one or more of thefollowing properties and/or functions, including, being infectious,begin replication competent, being productive, being produced atsubstantially the same copy number as in the absence of AAV Rep proteinexpression, being capable of site-specific integration, expressing Rep78protein and/or Rep68 protein at reduced levels compared to viruscontaining wild type AAV Rep inhibitory nucleotide sequence.

For example, the invention's viruses are infectious. Data hereindemonstrate productive infection that generated infectious virus that isreplication competent, using clone pAd/sRep78 (containing a scrambledRep78 sequence) and clone pAd/dRep78 (containing a deoptimized Rep78sequence), that formed CPE in transfected HEK 293 packaging cells(Example 3).

In another example, the invention's infectious viruses are replicationcompetent. Data herein demonstrate productive infection that generatedinfectious virus that is replication competent, using clone pAd/sRep78(containing a scrambled Rep78 sequence) and clone pAd/dRep78 (containinga deoptimized Rep78 sequence), that formed CPE in transfected HEK 293packaging cells (Example 3).

In a further example, the invention's replication competent viruses areproductive. Data herein demonstrate productive infection that generatedinfectious virus that is replication competent, using clone pAd/sRep78(containing a scrambled Rep78 sequence) and clone pAd/dRep78 (containinga deoptimized Rep78 sequence), that formed CPE in transfected HEK 293packaging cells (Example 3).

In another example, the copy number of the invention's viruses isproduced by a permissive cell at substantially the same copy number as athe copy number of a control virus that lacks expression of AAV Repprotein. Data herein demonstrate that the copy numbers of adenovirusAdsRep78 (containing a scrambled Rep78 sequence) and adenovirus AddRep78(containing a deoptimized Rep78 sequence) that were produced by HEK 293cells were substantially the same to each other, and to a controladenovirus Ad/AAVFVIII (which carries coagulation FVIII flanked by theAAV ITR in the absence of a Rep expression cassette) (Example 3).Furthermore, data herein also show that production of both adenovirusAdsRep78 and adenovirus AddRep78 could be scaled up with yieldscomparable to each other and to Ad/AAVFVIII (Example 3, Table 1).

In a further example, the invention's viruses are characterized bysite-specific integration of heterologous nucleotide sequences that theycontain into the adeno-associated virus integration site 1 (AAVS1)sequence of a host cell. In some embodiments, however, where delivery ofa heterologous nucleotide sequences is desired without site-specificintegration, this may be accomplished using AAV.

In one particular example, the invention's viruses express Rep78 proteinSEQ ID NO:04 (or a functional portion thereof) at a reduced levelcompared to the level expressed by a control hybrid virus that comprisesa wild type amino acid sequence SEQ ID NO:20 and/or SEQ ID NO:02 that isencoded by the wild type AAV Rep inhibitory nucleotide sequence listedas SEQ ID NO:17 and/or SEQ ID NO:01, respectively. For example, dataherein demonstrate that the deoptimized Rep78 nucleotide sequence, whichuses codons in underrepresented pairs, expressed Rep78 protein atreduced levels compared to wild type Rep78 nucleotide sequence due tocodon pair bias. Utilization of underrepresented codon pairs resulted inan ORF that is expressed at reduced levels.

i. rAAV

Thus, in one embodiment, the invention's virus is a “recombinantadeno-associated virus” (“rAAV”) containing one or more of theinvention's sequences. rAAV are widely used as gene transfer vehiclestoday, capable of long term extra chromosomal persistence in severaltissues. Production of rAAV [requires AAV ITR flanked transgene, AAV Repand Cap genes and helper virus. rAAV do not carry Rep due to sizeconstraints and worries about toxicity and are therefore incapable ofsite-specific integration. The only AAV elements retained are the AAVITRs which flank the transgene of interest. Currently, production ofrAAV requires co-transfection of multiple plasmid constructs, bearingthe AAV ITR flanked transgene construct, the AAV Rep-Cap codingsequences and the Adenovirus helper functions including E2, E4 and VAinto cell lines such as 293 which provide Ad E1 functions or infectionof producer cell lines carrying an integrated Rep Cap cassette and therAAV sequence, with a helper Ad.

The FDA has approved clinical trial, for rAAV vectors expressing theCystic Fibrosis Transmembrane Conductance Regulator (rAAV2-CFTR) (Flotte(1996) Hum. Gene Ther. 7:1145-1159; Flotte et al. (2003) Hum Gene Ther14(11):1079-1088), rAAV2-FIX for the delivery of coagulation FIX topatients with Hemophilia B (Manno et al. (2003) Blood 101(8):2963-2972;Manno et al. (2006) Nat Med 12(3):342-347), rAAV2-hRPE65v2 vectors forexpression of RPE65 in the treatment of Leber's congenital Amaurosis(LCA) (Bennicelli et al. (2008) Mol Ther 16(3):458-465), rAAV vectorCERE-20 expressing the neurotrophic factor Neurturin (NTN) to protectagainst the degeneration of dopaminergic neurons associated withParkinson's disease, and rAAV2 vector expressing al antitrypsin for atantitrypsin (AAT) deficiency associated lung disease (Brantly et al.(2009) PNAS 106(38):16363-16368; Mingozzi et al. (2009) Blood114(10):2077-2086).

ii. Hybrid Viruses

In another embodiment, the invention's virus is a hybrid virus thatcomprises the invention's AAV sequences and at least a portion of aheterologous virus genome sequence. In a particular embodiment, theheterologous virus genome sequence is gutted. The term “gutted” and“fully deleted” are used interchangeably in reference to a viral vector,and refer to a viral vector (e.g., naked DNA, plasmid, virus particle,etc.) that lacks all the coding sequences that are otherwise present ina wild type virus. Gutted vectors may contain non-coding viralsequences, e.g., terminal repeat sequences, and packaging sequences. Forexample, a gutted adenovirus vector lacks all adenovirus codingsequences and optionally contains adenovirus terminal repeat sequencesand/or packaging sequences (e.g., U.S. Pat. No. 5,994,132 to Chamberlainet al., U.S. Pat. No. 6,797,265 to Amalfitano et al., U.S. Pat. No.7,563,617 to Hearing et al., and U.S. Pat. No. 6,262,035 to Campbell etal.). Gutted vectors are preferred in certain embodiments since they donot express viral vector proteins and hence do not induce an adverseimmune or toxic response in a cell.

While not intending to limit the source or type of heterologous viruswhose genome sequences are included the invention's a hybrid viruses, inone embodiment, the heterologous virus is exemplified by, but notlimited to, adenovirus, herpes simplex virus, retrovirus, lentivirus,and baculovirus.

a. Hybrid Adenovirus

Thus in a particular embodiment, the hybrid virus comprises at least aportion of adenovirus genome. “Adenovirus” refers to a double-strandedDNA virus with a genome of approximately 36 Kb flanked by invertedterminal repeats. Adenovirus boasts of a wide tropism which can beincreased by replacement of the fiber knob carried on the icosahedralcapsid, responsible for contact with the host receptor, with that ofanother serotype. Adenovirus is of animal origin, such as avian, bovine,ovine, murine, porcine, canine, simian, and human origin. Avianadenoviruses are exemplified by serotypes 1 to 10 that are availablefrom the ATCC, such as, for example, the Phelps (ATCC VR 432), Fontes(ATCC VR 280), P7 A (ATCC VR 827), IBH 2A (ATCC VR 828), J2 A (ATCC VR829), T8 A (ATCC VR 830), and K 11 (ATCC VR 921) strains, or else thestrains designated as ATCC VR 831 to 835. Bovine adenoviruses areillustrated by those available from the ATCC (types 1 to 8) underreference numbers ATCC VR 313, 314, 639 642, 768 and 769. Ovineadenoviruses include the type 5 (ATCC VR 1343) or type 6 (ATCC VR 1340).Murine adenoviruses are exemplified by FL (ATCC VR 550) and E20308 (ATCCVR 528). Porcine adenovirus (5359) may also be used. adenoviruses ofcanine origin include all the strains of the CAV1 and CAV2 adenoviruses[for example, Manhattan strain or A26/61 (ATCC VR 800) strain]. Simianadenoviruses are also contemplated, and they include the adenoviruseswith the ATCC reference numbers VR 591 594, 941 943, and 195 203. Humanadenoviruses, of which there greater than fifty (50) serotypes are knownin the art, are also contemplated, including Ad2, Ad3, Ad4, Ad5, Ad11,Ad14, Ad7, Ad9, Ad12, Ad16, Ad17, Ad21, Ad26, Ad34, Ad35, Ad 40. Ad48,Ad49, Ad50 (e.g., U.S. Pat. No. 7,300,657 to Pau, U.S. Pat. No.7,468,181 to Vogels, and U.S. Pat. No. 6,136,594 to Dalemans). In onepreferred embodiment, the adenovirus is selected from adenovirus 2 (Ad2)and adenovirus 5 (Ad5).

Adenoviruses of animal origin can be obtained, for example, from strainsdeposited in collections, then amplified in competent cell lines andmodified as required (Hearing et al., U.S. Pat. No. 7,563,617).Techniques for producing, isolating and modifying adenoviruses have beendescribed in the literature and may be used within the scope of thepresent invention [Akli et al., Nature Genetics 3 (1993) 224;Stratford-Perricaudet et al., Human Gene Therapy 1 (1990) 241; patent EP185 573, Levrero et al., Gene 101 (1991) 195; Le Gal la Salle et al.,Science 259 (1993) 988; Roemer and Friedmann, Eur. J. Biochem. 208(1992) 211; Dobson et al., Neuron 5 (1990) 353; Chiocca et al., NewBiol. 2 (1990) 739; Miyanohara et al., New Biol. 4 (1992) 238; WO91/18088, WO 90/09441, WO 88/10311, WO 91/11525]. These differentviruses can then be modified, for example, by deletion, substitution,addition, etc. The complete genome sequences have been determined forhuman adenovirus type 2 (GenBank Accession No. J01917), human adenovirustype 5 (GenBank Accession No. M73260; and GenBank Accession No.NC-001406), human adenovirus type 12 (GenBank Accession No. NC-001460,X73487); human adenovirus type 17 (GenBank Accession No. NC-002067, AF108105), and human adenovirus type 40 (GenBank Accession No. L19443).

Adenovirus vectors have been used in gene therapy, particularly cancertherapy. e.g., vector ONYX015 (Heise C (1997) Nature Med. 3:639-645;Rothmann et al. (1998) J. Virol. 72:9470).

Adenovirus vectors have also been used as Ad-based vaccines formultiples diseases including Tuberculosis (Magalhaes et al. (2008) PLoSONE 3:e3790), malaria (Shott et al. (2008) Vaccine 26:2818-2823), rabies(Zhou et al. (2006) Mol Ther 14:662-672), influenza (Hoelscher et al.(2008) J Infect Dis 197:1185-1188), and leishmania (Resende et al.(2008) Vaccine 26:4585-4593).

“Adenovirus early gene regions” refers to nucleotide sequences which arederived from adenovirus and which are transcribed prior to replicationof the adenovirus genome. The early gene regions comprise E1a, E1b, E2a,E2b. E3 and E4. The E1a gene products are involved in transcriptionalregulation; the E1b gene products are involved in the shut-off of hostcell functions, mRNA transport, regulation of apoptosis induction, andinhibition of p53 tumor suppressor. E2a encodes a DNA-binding protein(DBP); E2b encodes the viral DNA polymerase and preterminal protein(pTP). The E3 gene products are not essential for viral growth in cellculture. The E4 regions encode regulatory proteins involved intranscriptional and post-transcriptional regulation of viral geneexpression; a subset of the E4 proteins are essential for viral growth.In contrast to the adenovirus early gene regions, the “adenovirus lategene regions” refers to adenovirus nucleotide sequences that aretranscribed after replication. The products of the late genes (e.g.,L1-5) are predominantly components of the virion as well as proteinsinvolved in the assembly of virions. The VA genes produce VA RNAs thatblock the host cell from shutting down viral protein synthesis. Theearly and late gene regions of adenovirus have been characterized (e.g.,in Ad2 genomic sequence; GenBank No. J01917).

In a more particular embodiment, the hybrid virus expresses a functionalAAV Rep protein (such as Rep78 and/or Rep68). Data herein demonstratethe ability of Ad/dRep78 and Ad/sRep78 to produce functional Rep78 asconfirmed by an excision assay which depends on Rep's ability to cleaveat a folded AAV ITR (Example 3).

In a more particular embodiment, the adenovirus lacks one or moreadenovirus early gene region. This is exemplified by adenovirus thatlacks adenovirus E1 gene coding sequence (Example 2), and/or lacksadenovirus E3 gene coding sequence. To illustrate, Example 4 shows thatin spite of expressing far higher levels of Rep78 than the tetracyclineinducible system, the invention's ΔE1ΔE3 adenoviruses carrying thehu1-sRep78 and hu1-dRep78 constructs were still capable of normal ratesof replication, and CPE.

b. Hybrid Herpes Simplex Virus

In another particular embodiment, the hybrid virus comprises at least aportion of herpes simplex virus, such as, without limitation, HSV-1 andHSV-2. “Herpes simplex virus” also referred to as “HSV”, is an envelopedvirus with a linear double stranded DNA (dsDNA) genome of 152 Kb,carrying 74 separate genes. The genome consists of 2 unique sequences,one longer than the other (U_(L) and U_(S)). Each of these sequences areflanked by inverted terminal repeat sequences—with U_(L) flanked byTerminal Repeat (TR_(L)) and Internal Repeat (IR_(L)) and U_(S) beingflanked by IR_(S) and TR_(S). Copies of an ‘a’ sequence carryingpackaging signals lie between the two IR_(S) and at each TR_(S). HSV isexemplified by HSV-1 and HSV-2, which are neurotropic pathogensassociated with a number of skin diseases from herpes labialis andherpes genitalis to the life threatening neonatal herpes and herpesencephalitis (Watanabe D (2010) Journal of Dermatological Science57(2):75-82).

Generic methods are known for producing oncolytic HSV based vectors andDNA vaccines. The two main types of HSV based vectors used are ampliconvectors and replication attenuated vectors.

Amplicon vectors are plasmids made up of repeated units of thetransgene, a packaging signal (pac) and an ISV origin of replication (R.R. S & N. F (1982) Cell 30:295). When introduced into a cell along withHSV helper functions, these amplicons replicate and are packaged as headto tail concatemers into infectious HSV virions. HSV Amplicons have beenused as DNA vaccines (Santos et al. (2006) Curr Gene Ther 6(3):383-392).

Replication attenuated HSV vectors have been used as oncolytic vectors.These vectors have deletions in genes (such as HSV-TK and HSV-RR) thatare required for replication of the virus in non-dividing cells and arethus capable of replication only in dividing (tumor) cells. Clinicaltrials (Phase I) for multiple HSV-1 derived oncolytic viruses forcolorectal carcinoma (Kemeny N, et al. (2006) Hum Gene Ther17(12):1214-1224), melanoma (MacKie et al. (2001) Lancet357(9255):525-526), breast cancer (Hu et al. (2006) Clin Cancer Res12(22):6737-6747), and malignant glioma (Markert et al. (2000) Gene Ther7(10):867-874), among others have been reported. All studies reportedsafety and toleration of HSV vectors.

In another particular embodiment, the hybrid virus comprises at least aportion of a retrovirus. “Retrovirus” is a small enveloped RNA virus,containing two identical single stranded positive sense RNA genomesenclosed in an enveloped capsid. Retroviruses have a genome flanked byLong Terminal Repeats (LTR) and 4 main genes gag, pol, pro and env.

c. Hybrid Lentivirus

In a further embodiment, the embodiment, the hybrid virus comprises atleast a portion of a lentivirus. “Lentiviruses” are a group of complexretroviruses that carry accessory gene which regulate and coordinateviral gene expression. Lentiviruses also differ from other retrovirusesin their ability to infect non-dividing cells as most other retrovirusesare incapable of traversing the nuclear membrane and can thus infectonly dividing cells where the nuclear membrane is dissolved.

d. Hybrid Baculovirus

In another embodiment, the hybrid virus comprises at least a portion ofa baculovirus. “Baculoviruses” are a family of large rod-shaped virusesthat can be divided to two genera: nucleopolyhedroviruses (NPV) andgranuloviruses (GV). While GVs contain only one nucleocapsid perenvelope, NPVs contain either single (SNPV) or multiple (MNPV)nucleocapsids per envelope. The enveloped virions are further occludedin granulin matrix in GVs and polyhedrin for NPVs. Moreover, GV haveonly single virion per granulin occlusion body while polyhedra containsmultiple embedded virions. Baculoviruses have very species-specifictropisms among the invertebrates with over 600 host species having beendescribed. They are not known to replicate in mammalian or othervertebrate animal cells. Baculoviruses contain circular double-strandedgenome ranging from 80-180 kbp.

Baculovirus expression in insect cells represents a robust method forproducing recombinant glycoproteins. Baculovirus-produced proteins haveseveral immunologic advantages over proteins derived from mammaliansources and are attractive candidates for therapeutic cancer vaccines(Betting et al., Enhanced immune stimulation by a therapeutic lymphomatumor antigen vaccine produced in insect cells involves mannose receptortargeting to antigen presenting cells. Vaccine. 2009 Jan. 7;27(2):250-9. Epub 2008 Nov. 8. PMID: 19000731).

E. Cells

The rAAV vector construct, and the complementary packaging geneconstructs can be implemented in this invention in a number of differentforms. Generic methods are known for introducing rAAV particles,plasmids, and stably transformed host cells into cells (e.g., packagingcell) either transiently or stably (Carter, U.S. Pat. No. 7,785,888).

Cells may be contacted with the recombinant viral vectors (e.g. rAAV)and AAV viral particles of the invention “in vivo,” “in vitro,” “exvivo,” and any combination thereof. As used herein, the term “in vitro”refers to an artificial environment and to processes or reactions thatoccur within an artificial environment. In vitro environments areexemplified, but not limited to, controlled laboratory conditions suchas test tubes, culture plates, culture wells, etc. The term “in vivo”refers to the natural environment (e.g., within an organism or a cell)and to processes or reactions that occur within that naturalenvironment. The term “ex vivo” refers to an environment wherein thecell is removed from, and manipulated outside, an organism and/ortissue.

A variety of different genetically altered cells can thus be used in thecontext of this invention. By way of illustration, a mammalian host cellmay be used with at least one intact copy of a stably integrated rAAVvector. An AAV packaging plasmid comprising at least an AAV rep geneoperably linked to a promoter can be used to supply replicationfunctions (U.S. Pat. No. 5,658,776). Alternatively, a stable mammaliancell line with an AAV rep gene operably linked to a promoter can be usedto supply replication functions (see, e.g., Trempe et al., U.S. Pat. No.5,837,484; Burstein et al., WO 98/27207; and Johnson et al., U.S. Pat.No. 5,658,785). The AAV cap gene, providing the encapsidation proteinsas described above, can be provided together with an AAV rep gene orseparately (see, e.g., the above-referenced applications and patents aswell as Allen et al. (WO 96/17947). Other combinations are possible(Carter, U.S. Pat. No. 7,785,888).

As is described in the art, and illustrated herein, genetic material canbe introduced into cells (such as mammalian “producer” cells for theproduction of rAAV) using any of a variety of means to transform ortransduce such cells. By way of illustration, such techniques include,but are not limited to, transfection with bacterial plasmids, infectionwith viral vectors, electroporation, calcium phosphate precipitation,and introduction using any of a variety of lipid-based compositions (aprocess often referred to as “lipofection”). Methods and compositionsfor performing these techniques have been described in the art and arewidely available (Carter, U.S. Pat. No. 7,785,888).

Once the host cell is provided with the requisite elements, the cell iscultured under conditions that are permissive for the replication ofAAV, to allow replication and packaging of the rAAV vector. rAAVparticles are then collected, and isolated from the cells used toprepare them (Carter, U.S. Pat. No. 7,785,888).

Selection of cells containing the invention's vectors and/or viruses maybe conducted by any technique in the art. For example, thepolynucleotide sequences used to alter the cell may be introducedsimultaneously with or operably linked to one or more detectable orselectable markers as is known in the art. By way of illustration, onecan employ a drug resistance gene as a selectable marker. Drug resistantcells can then be picked and grown, and then tested for expression ofthe desired sequence (i.e., a product of the heterologouspolynucleotide). Testing for acquisition, localization and/ormaintenance of an introduced polynucleotide can be performed using DNAhybridization-based techniques (such as Southern blotting and otherprocedures as known in the art). Testing for expression can be readilyperformed by Northern analysis of RNA extracted from the geneticallyaltered cells, or by indirect immunofluorescence for the correspondinggene product. Testing and confirmation of packaging capabilities andefficiencies can be obtained by introducing to the cell the remainingfunctional components of AAV and a helper virus, to test for productionof AAV particles. (Carter, U.S. Pat. No. 7,785,888).

In one embodiment to packaging rAAV vectors in an AAV particle, the rAAVvector sequence (i.e., the sequence flanked by AAV ITRs), and the AAVpackaging genes to be provided in trans, are introduced into the hostcell in separate bacterial plasmids (Carter, U.S. Pat. No. 7,785,888).

A second embodiment is to provide either the rAAV vector sequence, orthe AAV packaging genes, in the form of an episomal plasmid in amammalian cell used for AAV replication. See, for example, U.S. Pat. No.5,173,414 and Carter, U.S. Pat. No. 7,785,888.

F. Vaccines

The invention provides compositions (such as vaccines) comprising theinvention's AAV nucleotide and amino acid sequences, vectors, virusesand/or cells. In one embodiment, the composition is free of helpervirus. In another embodiment, the composition is a vaccine. The term“vaccine” refers to a pharmaceutically acceptable preparation that maybe administered to a host to induce a humoral immune response (includingeliciting a soluble antibody response) and/or cell-mediated immuneresponse (including eliciting a cytotoxic T lymphocyte (CTL) response).

In one embodiment, the composition further comprises a pharmaceuticallyacceptable compound such as diluent, carrier, excipient, and/oradjuvant.

The terms “pharmaceutically acceptable,” “pharmaceutical” and“physiologically tolerable” refer to a composition that containsmolecules that are capable of administration to or upon a subject andthat do not substantially produce an undesirable effect such as, forexample, adverse or allergic reactions, dizziness, gastric upset,toxicity and the like, when administered to a subject. Preferably also,the pharmaceutically acceptable molecule does not substantially reducethe activity of the invention's compositions. Pharmaceutical moleculesinclude, but are not limited to excipients and diluents. Vaccines maycontain pharmaceutically acceptable adjuvants, diluents, carriers,and/or excipients.

The term “adjuvant” as used herein refers to any compound which, wheninjected together with an antigen, non-specifically enhances the immuneresponse to that antigen. Exemplary adjuvants include Complete Freund'sAdjuvant, Incomplete Freund's Adjuvant, Gerbu adjuvant (GMDP; C.C.Biotech Corp.), RIBI fowl adjuvant (MPL; RIBI Immunochemical Research,Inc.), potassium alum, aluminum phosphate, aluminum hydroxide, QS21(Cambridge Biotech), Titer Max adjuvant (CytRx), and Quil A adjuvant.Other compounds that may have adjuvant properties include binders suchas carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose,or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, Primogel,corn starch and the like; lubricants such as magnesium stearate orSterotex; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin, a flavoring agent such as peppermint,methyl salicylate or orange flavoring, and a coloring agent.

Exemplary “diluents” include water, physiological saline solution, humanserum albumin, oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents, antibacterial agents such as benzylalcohol, antioxidants such as ascorbic acid or sodium bisulphite,chelating agents such as ethylene diamine-tetra-acetic acid, bufferssuch as acetates, citrates or phosphates and agents for adjusting theosmolarity, such as sodium chloride or dextrose.

Exemplary “carriers” include liquid carriers (such as water, saline,culture medium, saline, aqueous dextrose, and glycols) and solidcarriers (such as carbohydrates exemplified by starch, glucose, lactose,sucrose, and dextrans, anti-oxidants exemplified by ascorbic acid andglutathione, and hydrolyzed proteins.

The term “excipient” refers herein to any inert substance (e.g., gumarabic, syrup, lanolin, starch, etc.) that forms a vehicle for deliveryof an antigen. The term excipient includes substances that, in thepresence of sufficient liquid, impart to a composition the adhesivequality needed for the preparation of pills or tablets.

G. Exemplary Applications of the Invention's Compositions forIdentification of Functional Portions of Wild Type AAV Rep InhibitoryNucleotide Sequences

The invention's compositions may be used to identity functional portionsof the wild type AAV Rep inhibitory nucleotide sequence listed as SEQ IDNO:17 and/or SEQ ID NO:01, i.e., portions that reduce replication and/orinfection and/or productive infection by a virus.

In a first embodiment, the method utilizes a scrambled AAV Repinhibitory nucleotide sequence (exemplified by, but not limited to, SEQID NO:18 and/or SEQ ID NO:07 and/or portion thereof). In a secondembodiment, the method utilizes a portion of the wild type AAV Repinhibitory nucleotide sequence listed as SEQ ID NO:17 and/or SEQ IDNO:01.

i. Methods Employing Scrambled Sequences

Thus, in a first embodiment, the invention provides methods fordetecting a sequence that reduces replication and/or infection and/orproductive infection by a virus, comprising a) providing i) a firstexpression vector comprising a first nucleotide sequence comprising ascrambled polynucleotide sequence encoding a portion of wild type AAVRep inhibitory amino acid sequence listed as SEQ ID NO:20 and/or SEQ IDNO:02, ii) a second expression vector comprising a second nucleotidesequence, wherein the second nucleotide sequence is produced bysubstituting a portion of the scrambled polynucleotide sequence with acorresponding portion of wild type AAV Rep inhibitory nucleotidesequence listed as SEQ ID NO:17 and/or SEQ ID NO:01, and iii) a hostcell that is permissive for the virus, b) transfecting i) the firstexpression vector into the permissive cell under conditions to produce afirst virus that comprises a first amino acid sequence encoded by thefirst nucleotide sequence, and ii) the second expression vector into thepermissive cell under conditions to produce a second virus thatcomprises a second amino acid sequence encoded by the second nucleotidesequence, and c) detecting reduced replication and/or infection and/orproductive infection of the permissive cell by the second virus comparedto the first virus, wherein the detecting indicates that the portion ofwild type AAV Rep inhibitory nucleotide sequence reduces replicationand/or infection and/or productive infection by the virus. While notnecessary, it may be desirable that the detecting step comprises, afterthe transfecting step, isolating one or more of i) the first virus, andii) the second virus.

The invention's methods are exemplified by S (wt3) Rep (FIG. 3A)produced in Example 5, with S (wt3) Rep in which a 564 bp portion of ascrambled Rep78 ORF was substituted with bp 1623 to bp 2186 bp of theAAV2 genome corresponding to ˜C-terminal third of the wild type Rep ORF,and which showed no replication, thus demonstrating that an AAV Repinhibitory nucleotide sequence was localized within the 3′ 564 basepairs, in the region encompassing bp 1623 to bp 2186 of the AAV2 genome.A similar approach was used with respect to Ad/Rep I (FIG. 3B), tofurther narrow the location of the AAV Rep inhibitory nucleotidesequence to the 135-bp sequence from bp 1782 to bp 1916 of the AAV2genome, FIG. 4A-H (Example 5).

ii. Methods Employing Wild Type Sequences

In a second embodiment, the invention provides methods for detecting asequence that reduces replication and/or infection and/or productiveinfection by a virus, comprising a) providing i) a first expressionvector comprising a first nucleotide sequence comprising a portion ofwild type AAV Rep inhibitory nucleotide sequence listed as SEQ ID NO:17and/or SEQ ID NO:01, ii) a second expression vector comprising a secondnucleotide sequence, wherein the second nucleotide sequence is producedby substituting the portion of the wild type AAV Rep inhibitorynucleotide sequence with a scrambled polynucleotide sequence encodingthe portion of the wild type AAV Rep inhibitory nucleotide sequence, andiii) a host cell that is permissive for the virus, b) transfecting i)the first expression vector into the permissive cell under conditions toproduce a first virus that comprises a first amino acid sequence encodedby the first nucleotide sequence, and ii) the second expression vectorinto the permissive cell under conditions to produce a second virus thatcomprises a second amino acid sequence encoded by the second nucleotidesequence, c) detecting increased replication and/or infection and/orproductive infection of the permissive cell by the second virus comparedto the first virus, wherein the detecting indicates that the portion ofthe wild type AAV Rep inhibitory nucleotide sequence reduces replicationand/or infection and/or productive infection by the virus.

While not necessary, it may be desirable that the detecting stepcomprises, after the transfecting step, isolating one or more of i) thefirst virus, and ii) the second virus.

The invention's methods are exemplified by the virus produced in Example5, with S (wt1,2) Rep (FIG. 3A) in which a 555 bp portion encompassingbp 1623 to bp 2186 bp of the AAV2 genome corresponding to ˜C-terminalthird of the wild type Rep ORF was substituted with a correspondingscrambled sequence, and which showed that modification of these 564 bpportion of the wild type sequence with scrambled Rep sequences, alone,was sufficient to lift inhibition of productive infection, and allowreplication of the adenovirus carrying it, comparable to Ad/sRep78 thatcontained an entirely scrambled sequence of wild type AAV Rep78.

H. Exemplary Applications of the Invention's Compositions for Generationof Viruses

The invention provides methods for producing a recombinantadeno-associated virus (rAAV) particle, comprising a) providing anexpression vector comprising one or more of the nucleotide sequencesdescribed herein (e.g., a nucleotide sequence encoding a chimericprotein, a) wherein the encoded chimeric protein i) comprises at least aportion of wild type AAV Rep inhibitory amino acid sequence listed asSEQ ID NO:20 (i.e., encoded by the 564-bp DNA sequence from bp 1623 tobp 2186 of the AAV2 genome) and/or SEQ ID NO:02 (i.e., encoded by the135-bp DNA sequence from bp 1782 to bp 1916 of the AAV2 genome, FIG. 4B), and ii) has Rep-mediated nuclease activity, and b) wherein therecombinant nucleotide sequence comprises a scrambled polynucleotidesequence encoding the wild type AAV Rep inhibitory amino acid sequencelisted as SEQ ID NO:20 and/or SEQ ID NO:02), and/or portion thereof, b)providing an adeno-associated virus (AAV) packaging cell, and c)transfecting the packaging cell with the expression vector to produce arecombinant adeno-associated virus (rAAV).

In one embodiment, the method further comprises detecting the presenceof the produced recombinant adeno-associated virus (rAAV).

In a further embodiment, the method further comprises isolating therecombinant adeno-associated virus (rAAV).

In a particular embodiment, the method does not include (i.e., lacks)the step of transfecting the packaging cell with a helper virus [thishighlights advantage of using ONLY one virus for expression of allgenetic elements] [this highlights one advantage of the invention's AAVsequences, vectors, rAAV particles, and hybrid viruses (e.g., Ad/AAV) inthat they tolerate the inclusion of all genetic elements in a singlevirus for the purpose of safely integrating a transgene into a saferegion of the human genome, which provides a safer alternative tocurrent approaches that use retroviruses and lentiviruses in genereplacement strategies.

I. Exemplary Applications of the Invention's Compositions for Expressionof Nucleotide Sequences (e.g., in Gene Therapy and/or VaccineApplications)

The invention's compositions (such as AAV nucleotide sequences, vectors,viruses (e.g., rAAV particles, and hybrid AAV particles such as Ad/AAV),and/or cells) are useful in several contexts, including, but not limitedto, gene therapy and vaccine applications. Thus, in one embodiment, theinvention provides methods for reducing one or more symptoms of diseasein a mammalian subject, comprising administering a therapeuticallyeffective amount of one or more of the invention's compositions (such asAAV nucleotide sequences, vectors, viruses (e.g., rAAV particles, andhybrid AAV particles such as Ad/AAV), and/or cells) to a mammaliansubject in need of therapy. In particular embodiments the invention'scompositions contain the recombinant nucleotide sequences describedherein, and further contain a heterologous polynucleotide sequence(optionally operably linked to AAV ITR). The mammalian subject includes,without limitation, a subject that has a disease and a subject at riskof disease.

In one example, one or more of the invention's compositions (such as AAVnucleotide sequences, vectors, viruses (e.g., rAAV particles, and hybridAAV particles such as Ad/AAV), and/or cells) are useful in gene therapyapplications. In these applications, it is desirable that theheterologous polynucleotide sequence comprises a therapeutic sequence.

In another example, the invention's compositions (such as AAV nucleotidesequences, vectors, viruses (e.g., rAAV particles, and hybrid AAVparticles such as Ad/AAV), and/or cells) are useful in vaccineapplications. In these applications, it is desirable that heterologouspolynucleotide sequence encodes an antigen polypeptide. While notnecessary, in one embodiment the method further comprises detecting theimmune response to the antigen polypeptide.

The invention's compositions (such as AAV nucleotide sequences, vectors,and/or viruses are administered in a therapeutic amount. The terms“therapeutic amount,” “pharmaceutically effective amount,”“therapeutically effective amount,” “biologically effective amount,” and“protective amount” are used interchangeably herein to refer to anamount that is sufficient to achieve a desired result, whetherquantitative and/or qualitative. In particular, a therapeutic amount isthat amount that delays, reduces, palliates, ameliorates, stabilizes,prevents and/or reverses one or more symptoms of the disease compared toin the absence of the composition of interest. Examples include, withoutlimitation, tumor size and/or tumor number in cancer disease, glucoselevels in blood and/or urine in diabetes, standard biochemical kidneyfunction tests in kidney disease, etc. The terms also include, inanother embodiment, an amount of the composition that reduces infectionby a pathogen (e.g., HIV, malaria parasite, Pseudomonas species),regardless of whether disease symptoms are altered (i.e., increased orreduced).

In vaccine applications, the invention's compositions are preferablyadministered in an immunologically effective amount. In one embodiment,“immunogenically effective amount” and “immunologically-effectiveamount” refer to that amount of a molecule that elicits and/or increasesproduction of an immune response (including production of specificantibodies and/or induction of a cytotoxic T lymphocyte (CTL) response)in a host upon vaccination.

Specific “dosages” can be readily determined by clinical trials anddepend, for example, on the route of administration, patient weight(e.g. milligrams of drug per kg body weight). The term “delaying”symptoms refers to increasing the time period between exposure to theimmunogen or virus and the onset of one or more symptoms of theexposure. The term “eliminating” symptoms refers to 100% reduction ofone or more symptoms of exposure to the immunogen or virus.

As used herein, the actual amount, i.e., “dosage,” encompassed by theterm “pharmaceutically effective amount,” “therapeutically effectiveamount,” “immunologically effective,” and “protective amount” willdepend on the route of administration, the type of subject beingtreated, and the physical characteristics of the specific subject underconsideration. These factors and their relationship to determining thisamount are well known to skilled practitioners in the medical,veterinary, and other related arts. This amount and the method ofadministration can be tailored to achieve optimal efficacy but willdepend on such factors as weight, diet, concurrent medication and otherfactors that those skilled in the art will recognize. The dosage amountand frequency are selected to create an effective level of the compoundwithout substantially harmful effects.]

An effective amount of recombinant viral vector, such as rAAV and/orhybrid viruses is administered, depending on the objectives oftreatment. An effective amount may be given in single or multiple doses.Where a low percentage of transduction can achieve a therapeutic effect,the objective of treatment is generally to meet or exceed this level oftransduction. In some instances, this level of transduction can beachieved by transduction of only about 1 to 5% of the target cells, butis more typically 20% of the cells of the desired tissue type, usuallyat least about 50%, preferably at least about 80%, more preferably atleast about 95%, and even more preferably at least about 99% of thecells of the desired tissue type (Carter, U.S. Pat. No. 7,785,888).

As a guide, the number of virus particles administered per injectionwill generally be between 1×10⁶ and 1×10¹⁴ particles, preferably,between 1×10⁷ and 1×10¹³ particles, more preferably 1×10⁹ and 1×10¹²particles and even more preferably about 1×10¹¹ particles (Carter, U.S.Pat. No. 7,785,888).

The number of virus particles administered per intramuscular injectionand per intravenous administration, for example, will generally be atleast about 1×10¹⁰, and is more typically 5×10¹⁰, 1×10¹¹, 5×10¹¹,1×10¹², 5×10¹² and on some occasions 1×10¹³ particles (Carter, U.S. Pat.No. 7,785,888).

In one embodiment, the invention's methods further comprise the step ofdetecting the presence of at least a portion of the vector in a cell ofthe treated subject.

In a further embodiment, it may be desirable to confirm theeffectiveness of delivery of the invention's compositions (such as AAVnucleotide sequences, vectors, and/or viruses) to target cells. This canbe monitored by several criteria. For example, samples removed by biopsyor surgical excision can be analyzed by in situ hybridization, PCRamplification using vector-specific probes, and/or RNAse protection todetect viral DNA and/or viral mRNA, such as rAAV DNA or RNA. Also, forexample, harvested tissue, joint fluid and/or serum samples can bemonitored for the presence of a protein product encoded by therecombinant viral vector with immunoassays, including, but not limitedto, immunoblotting, immunoprecipitation, immunohistology and/orimmunofluorescent cell counting, or with function-based bioassays.Examples of such assays are known in the art (Carter. U.S. Pat. No.7,785,888).

Administration of the invention's compositions (such as AAV nucleotidesequences, vectors, and/or viruses to a mammalian subject, so as tointroduce a sequence of interest into a mammalian cell and/or express agene product of interest in a mammalian cell) can be accomplished inseveral ways, that include, but are not limited to, intramuscularadministration, intradermal administration, intravenous administration,subcutaneous administration, aerosol administration, oraladministration, and/or sub-lingual administration. Administrationincludes direct injection of the composition(s) to a tissue oranatomical site. Injection can be, for example, intra-arterial,intravenous, intramuscular or intra-articular. Administration may beparenteral, oral, intraperitoneal, intranasal, topical, etc. Parenteralroutes of administration include, for example, subcutaneous,intravenous, intramuscular, intrasternal injection, and infusion routes.Methods of transducing cells of blood vessels are described, forexample, in PCT US97/103134.

Another preferred mode of administration of compositions of theinvention is through naso-pharyngeal and pulmonary routes. Theseinclude, but are not limited to, inhalation, transbronchial andtransalveolar routes. The invention includes compositions suitable foradministration by inhalation including, but not limited to, varioustypes of aerosols and powder forms. Devices suitable for administrationof compositions by inhalation include, but are not limited to, atomizersand vaporizers (Carter, U.S. Pat. No. 7,785,888).

The compositions of the invention may be administered before,concomitantly with, and/or after manifestation of one or more symptomsof a disease or condition. Also, the invention's compositions may beadministered before, concomitantly with, and/or after administration ofanother type of drug or therapeutic procedure (e.g., surgery, radiation,etc.). For example, in the case of pathogen infection, the invention'scompounds may be administered before, concomitantly with, and/or afteradministration of antibiotics and/or antivirals.

Administration may be in vivo and/or or ex vivo to deliver a transgeneto an individual, preferably a mammal. Such methods and techniques areknown in the art. See, for example, U.S. Pat. No. 5,399,346. Generally,cells are removed from an individual, transduced by recombinant viralvectors, such as rAAV vectors, in vitro, and the transduced cells arethen reintroduced into the individual. Cell suitable for ex vivodelivery are known to those skilled in the art and include, for example,various types of stem cells (Carter, U.S. Pat. No. 7,785,888).

The selection of a particular composition, dosage regimen (i.e., dose,timing and repetition) and route of administration will depend on anumber of different factors, including, but not limited to, thesubject's medical history and features of the condition and the subjectbeing treated, and may be determined empirically (Carter, U.S. Pat. No.7,785,888).

In one embodiment of the invention, methods for identifying a phenotypeassociated with expression of a coding sequence of a recombinant viralvector of the invention are provided, comprising subjecting host cellscontaining a recombinant viral vector of the invention to conditionswhich allow expression; comparing a phenotype of these expressing cellsto a phenotype of cells which lack the recombinant viral vector; whereina phenotypic difference indicates a phenotype associated with expressionof the coding sequence. In other embodiments, phenotypic screening isaccomplished by contacting a host cell with a recombinant viral vectordescribed herein under conditions that allow uptake of the vector;assaying the cell for expression of the heterologous coding region ofthe vector; comparing a phenotype of the cell expressing theheterologous coding region with a phenotype of a cell that lacks thevector. A phenotypic difference indicates that the phenotype of the cellexpression the heterologous sequence is a phenotype associated withexpression of the coding region. Such phenotypic characteristics couldin turn provide valuable information regarding function(s) of the codingsequence, as well as its potential role in health or contributing todisease states, and as a useful drug target (Carter, U.S. Pat. No.7,785,888).

EXPERIMENTAL

The following examples serve to illustrate certain embodiments andaspects of the present invention and are not to be construed as limitingthe scope thereof.

Example 1

Materials and Methods

De Novo Synthesized Constructs:

All de novo synthesized sequences were synthesized and cloned into pUC13by GenScript USA. Scrambled Rep, Deoptimized Rep, were designed using aprogram previously described (13). The sequences were designed withunique restriction sites SbfI and SwaI flanking them as well as a uniqueR.E Afe1 at bp 661 of the AAV2 genome to allow ease of manipulation. Anaturally occurring R.E BstBI site that occurs at bp 1623 of of the AAV2genome was retained.

Rep78 Design 1, 11, 111 and IV was designed using algorithms describedin the art (Coleman et al., Science 320:1784 (2008). S (wt3) Repcontains wild type Rep sequences from AAV2 bp 1623 to 2186. S (wt1,2)Rep contains scrambled Rep sequences from AAV2 bp 1623 to 2186. Theconstructs shown in FIG. 3A-C delimit the 135 bp interval from bp 1782to bp 1916 of the AAV2 genome.

Plasmids and Cloning:

For detection of expression levels from wild-type, scrambled andDeoptimized Rep ORFs, each ORF was PCR amplified without the stop codonand cloned into pCMV-3Tag3A (Stratagene, USA) under the CMV minimalpromoter, with a 3×C terminal flag tag. The first step in theconstruction of all viral constructs was the cloning of the requiredtransgene into a shuttle vector. All shuttle vectors were derived frompAd/AAV-EGFP-Neo (18).

pTROTS-wt was constructed by PCR amplification and stepwise cloning ofthe TRE-pTK-Rep78 cassette and the pCMV-tTS cassette from plasmid pΔ28(a gift from Dr. Mavilio, Italy) in place of the pCMV-EGFP-Neo cassettein pAd/AAV-EGFP-Neo. The Rep78 ORF was flanked by a unique SbfI siteupstream and SwaI site downstream, and these sites were usedsubsequently to swap in the Scrambled and Deoptimized Rep ORFs frompUCScr and pUCDeopt in place of wild-type, to construct pTROTS-Scr andpTROTS-Deopt.

For localization of the sequence signal, various sections of theScrambled ORF were replaced with the corresponding wild-type sequence inframe. AAV2 bp 321 to bp 983 of the wild type Rep sequence was amplifiedfrom pTROTS-wt by PCR and swapped into pTROTS-Scr replacing thecorresponding Scrambled sequence. Since BstBI was not unique withinpTROTS-Scrambled, pTROTS Scr (wt2) and pTROTS Scr (wt3) had to beconstructed in two steps. The AAV2 bp 984 to bp 1623 of wild-type Rep(fragment wt2) was PCR amplified from pTROTS-WT and cloned into thepUCScr, making pUCScr(wt2). This entire Rep cassette was excised out ofby restriction digestion with R.E SbfI and SwaI and inserted into thepTROTS backbone, making pTROTS-Scr(wt2). Similarly, pUCScr(wt3) wasconstructed by excision of AAV2 bp 1623 to bp 2186 of wild-type Rep(wt3) from pTROTS-wt and ligation into similarly digested pUCScr,resulting in pUCScr(wt3). The entire Rep coding ORF was then swappedinto pTROTS backbone, resulting in pTROTS-Scr(wt3). Similarly, shuttlevectors for construction of Ad/Rep78-1 to Ad/Rep78-IV were constructedby excision of the entire Rep coding ORF from the pUC13 clone by doubledigestion with R.Es SbfI and SwaI and insertion into the sites withinpTROTS-wt, replacing the wtRep78 ORF.

phu1Scr and phu1Deopt have the Scrambled and Deoptimized Rep constructsrespectively, expressed under an hu1 promoter. A left end shuttle vectorcarrying the hu1 promoter and SV40 poly A was constructed by digestionof the cassette from pcDNA3-hu1polyA (17) by flanking XbaI restrictionsites and cloned in place of the pCMV-EGFP-Neo cassette inpAd/AAV-EGFP-Neo, generating pITR-packaging-hu1 polyA-3330. Unique BglIIand HindIII restriction enzyme sites lay between the hu1 promoter andthe poly A sequence. The Scrambled Rep78 ORF was PCR amplified frompUCScr and inserted into the BglII site in the vector, and theorientation checked by sequencing. phu1Deopt was constructed by PCRamplification of the Deoptimized ORF from pUCDeopt and introduced intothe BglII HindIII double digested vector backbone.

For the construction of a shuttle vector for Ad/AAVFVIII, 2 copies ofthe AAV integration efficiency element was originally cloned upstream ofthe AAV ITR in pAd/AAVCMV-EGFP-Neo. The IRES-EYFP cassette frompIRES-EYFP (Stratagene, USA) was PCR amplified and cloned downstream ofthe 5.6 Kb pPF4-FVIII cassette in pBSPF4FVIII (19). This pPF4-FVIII-EYFPcassette was then excised out by NotI digestion and inserted intopAd/AAV CMV-EGFP-Neo and transformed into Max Efficiency Stbl2 cells(Imitrogen, USA). Further details of plasmid construction available onrequest. All restriction enzymes used were from NEB USA. PfuUltraHigh-Fidelity DNA Polymerase (Stratagene, USA) was used for all PCRamplification steps.

Virus Construction:

All viruses were constructed by homologous recombination in BJ5183 cells(Stratagene, USA), between the shuttle vectors and pTG3602 ΔE3 F5/35.pTG3602 (20) contains the intact WT Adenoviral genome and was obtainedfrom Transgene, S.I (Strasbourg, France). Details of cloning from Pat.

Positive clones were transformed into DH5α cells to scale up production.Ad/AAV fVIII alone was electroporated into SURE electroporationcompetent cells (Stratagene, USA) as DH5α cells were found to beunsuitable for maintenance of the intact AAV ITR. Transformed cloneswere confirmed by restriction digestion.

5 μg of viral DNA was linearized and used to transfect 80% confluent 6cm plates of HEK 293 packaging cells using Fugene 6 (Roche, USA),following manufacturer's directions. At day 10, cells were lysed byfreeze thaw and lysate used to infect fresh cells for the development ofcytopathic effect (CPE).

Excision Assay:

6 cm plates of 293 cells were co-infected with Ad/AAVFVIII and eitherAd/sRep78 or Ad/dRep78 at an MOI of 50 each. 1 hr after infection, cellswere induced with doxycycline. Cells infected with Ad/AAVFVIII onlyserved as the negative control, while C12 cells co-infected withAd/AAVFVIII and Wild-type Adenovirus served as a positive control. Inthe case of Rep expressing plasmids, 293 cells in 6 well plates weretransfected with 2 μg of the plasmid using Fugene 6 (Roche, USA)following manufacturer's instructions, 48 hours before infection withAd/AAVFVIII.

48 hours post infection, cells were lysed and Hirt extrachromosomal DNAisolated. One quarter of the total DNA prep was run out on a 0.8%agarose gel and transferred onto nylon membranes (Roche, USA). Thepresence of excision products was detected by Southern blotting,following standard procedure. Non radioactive Digoxigenin labeled probes(Roche, USA) which recognized a ˜700 bp sequence at the junction ofpPF4-FVIII were used.

Viral Replication Assay:

To compare the ability of viruses carrying Rep to replicate, a modifiedDpnI viral replication assay was performed. 293 cells in 6 well plateswere transfected with linearized viral constructs. At various timepoints, cells were washed and low molecular weight (Hirt)extrachromosomal DNA isolated. 100 ng DNA was digested with DpnIovernight. DpnI requires dam methylated substrates. As the transfectedviral DNA is of bacterial origin, DpnI digestion ensures that viral DNAdetected is only replicated DNA. Southern blot analysis was completedusing non-radioactive Digoxigenin labeled probes using the DIG Easy Hybkit (Roche) following manufacturer's protocol.

Immunoblotting: HeLa cells in 6 well plates were transfected with equalamounts of pCMV-wtRep78-flag, pCMV-sRep78r-flag, or pCMV-dRep78-flagusing fugene6 (Roche, USA) following manufacturer's instructions. 48hours post transfection, cells were lysed using NP40 lysis buffer (50 mMTris.HCl pH8.0, 150 mM NaCl, 1% NP-40). Equal amounts of reduced,denatured protein was separated on a 4-15% polyacrylamide gel (Biorad,USA) and transferred onto a nitrocellulose membrane. Membranes wereblocked in 3% milkfat and incubated with primary antibody (mousemonoclonal anti-flag M2 (Sigma, USA) or mouse monoclonal anti-GAPDHMAB374 (Millipore, USA)) for 1 hour at room temperature, followed byincubation for 1 hour at RT with the secondary antibody (ECL Anti-mouseIgG Horseradish peroxidase linked F(ab′)₂ fragment from sheep (GEHealthcare, UK)). Detection was performed using the Pierce ECL WesternBlotting substrate (Thermo Scientific, USA) following standard protocol.

Example 2

Modification of Rep ORF:

To construct a first generation Ad carrying Rep78, the inventorsexpressed Rep under a tightly regulated tetracycline inducible promoterwithin an ΔE1ΔE1 F5/35 Adenovirus. The fiber knob of this Ad5 wasreplaced with that of Ad35 to allow it to infect hematopoietic cells(3). The tetracycline inducible Rep78 expression cassette, has beenpreviously used successfully for the construction of a helper dependentAdenovirus carrying Rep78 (9). Surprisingly, the inventors found thatthe same construct on an E1 deleted backbone was incapable ofreplication, showing no signs of viral growth in spite of multiplepassages in HEK 293 packaging cells. The inventors hypothesized that thereplicative functions provided by multiple helper virus genomes in transto the helper dependent virus allowed replication, whereas a singlegenome carrying both Adenoviral genes and the Rep expression constructwas unable to escape Rep's inhibitory effect.

To elucidate the relative contribution of the sequence of the Rep ORFand Rep protein levels on this apparently cis acting inhibitory effect,the inventors modified the 1865 bp Rep78 nucleotide sequence in silico.The inventors utilized an algorithm (13) that allowed us to modify thenucleotide sequence of Rep78 by about 20% to about 30% without affectingthe amino acid sequence encoded, using synonymous codons (Sequencealignments in FIG. 10A-F). Two modified Rep sequences, Scrambled andDeoptimized, were designed and synthesized de novo.

The Scrambled sequence randomly mixes synonymous codons, resulting in anucleotide sequence that differs from the wild-type sequence by about30%. The protein expressed from this ORF is identical to wild-typeRep78. This sequence aims to disrupt any sequence specific signal,without affecting Rep78 expression levels.

Within the Deoptimized sequence, synonymous codons are specificallypaired into under-utilized codon pairs (FIG. 1a ). Synonymous codons canbe paired in multiple ways to encode the same 2 adjacent amino acids.However, in nature a strong codon pair bias is found to exist, resultingin the disproportionate representation of some codon pairs over others(14). This codon pair bias is independent of codon frequency and isfound to affect translation rates. Utilization of under-representedcodon pairs such as those in Deoptimized sequences, therefore, resultsin an ORF that is expressed at lower levels due to inefficienttranslation (15). Thus, the Deoptimized Rep construct not only differsfrom the nucleotide sequence of wild-type Rep by 20%, presumablydisrupting any sequence specific signal, but also further reduces levelsof Rep78 expression from the tetracycline (Tet) inducible promoter.Confirmation of Deoptimized Rep's reduced ability to express protein wasobtained by immunoblot analysis of transfected C-terminal flag taggedconstructs, expressed under pCMV. Protein levels from Wild-type andScrambled Rep were found to be comparable to each other and roughlydouble that of Deoptimized (FIGS. 1B and 1C)

Example 3 Modification of Rep ORF Allows Replication of Adenovirus:

The Scrambled and Deoptimized Rep constructs were cloned downstream ofthe tetracycline inducible promoter, in place of the wild-type Rep ORF,within the fiber modified first generation Adenovirus genome, generatinginfectious clones pAd/sRep78 and pAd/dRep78 (FIG. 2A). These viralconstructs were linearized and transfected into HEK 293 packaging cellsand passaged every 10 days onto fresh cells, until the development ofCPE was observed. As mentioned earlier, no signs of viral replicationcould be observed with pAd/WTRep78 even with passaging up to 50 days.However, complete CPE was observed with both pAd/sRep78 and pAd/dRep78within a total of 15 days from transfection. Production of both virusescould be scaled up with infectious virus yields comparable to each otherand to Ad/AAVFVIII (Table 1), proving a clear role for the sequence ofRep in the inhibition of Adenoviral replication.

TABLE I Viral titers Virus Titer^(†) (PFU/mL × 10⁸) Ad/sRep 9.50 + 0.50Ad/dRep 9.50 + 0.25 Ad/s(wt1) Rep 8.13 + 0.12 Ad/s(wt2) Rep 8.75 + 0.25Ad/s(wt1, 2) Rep 8.13 + 0.12 Ad/Rep II 11.5 + 0.75 Ad/Rep III 10.4 +0.37 Ad/Rep IV 9.38 + 0.12 Ad/HU1-1/sRep 7.50 + 0.75 Ad/HU1-1/dRep8.00 + 0.50 Ad/AAV/PF4/BDD 10.5 + 0.50 ^(†)Titers were calculated byserial dilution and plaque assay in HEK 293 packaging cells, and arereported in plaque-forming units (PFU)/mL as the mean + SEM from twodistinct determinations

TABLE 2Putative transcription factor binding sites unique to wtRep78 bp1461-1596identified by TESS (Transcription Element Search System) (326) BeginTranscription Factor bp no. Sequence 1T00111 c-Ets-1 T00112 c-Ets-1 T00114 c-Ets-1 54 97 SMGGAWGYT00115 c-Ets-1 68 T00684 PEA3 T00685 PEA3 T00686 PEA3 2T00506 MEF1 T00519 Myf-3 T00524 MyoD 69 GTCAGTTGT00525 MyoD T00526 MyoD T00527 MyoD T01128 MyoD 3_00000 ASF-1 _00000 MSN4 _00000 deltaCREB 88 ACGTCA 4T00049 ATF T00050 atfl T00132 c-Jun T00163 88 ACGTCACREB T00164 CREB T00166 deltaCREB T00167CRE-BP1 T00846 TREB-1 T00942 EivF T01095 ATF3 5 T00163 CREB 33 TGACG 6T00051 ATF T00052 ATF-a T00053 ATF-adelta 33 TGACGYMRT00054 ATF-like T00442 47-kDa CRE bind. prot. T00968 ATF-1 7_00000 LRF-1 88 ACGTCA 8 _00000 ATF-1 88 ACGTCA 9T00134 c-Jun T00893 v-Jun 66 CGAGTCAG 10T00074 gammaCAC1 T00075 gammaCAC2 3 GGGTG T00077 CACCC-binding factor 11_00000 MIG1 27 CCCCAG 12 T00765 SRF (504 AA) 41 ATATA 13 _00000 RC2 18AAGACC 14 _00000 GCN4 67 GAGTCA 15 _00000 B-factor 42 TATAAGT 16T00386 HSTF 14 AGAAA 17 T00029 AP-1 90 GTCA 18 _00000 HBP-1 88 ACGTCA 19T00140 c-Myc 71 CAGTTG 20 T00029 AP-1 T00123 c-Fos T00133 c-Jun T0016733 TGACGCA CRE-BPI T00989 CREB T01313 ATF3 T02361 CREBbeta 21T00182 DBF4 T00270 ETF T00530 NC1 T00794 42 TATAATBP T00798 TBP T00817 TFIIA T00818 TFIIBT00820 TFIID T00835 TMF T00862 UBP-1T02216 TFIIA-alpha/beta precursor (majorT02216T02217 TFIIA-alpha/beta precursor (minorT02217 T02224 TFIIA-gamma 22T00074 gammaCAC1 T00075 gammaCAC2 59 GGGTG T00077 CACCC-binding factor23 T00321 GCN4 67 GAGTCA 24 T00029 AP-1 69 GTCA 25 T00029 AP-1 23CCGCCCCC 26 T00878 USF2 T02115 USF2 T02377 USF2b 33 TGACGCA 27T00422 IRF1 T00425 IRF-2 45 AAGTGA 28 T00968 ATF-1 88 ACGTCA 29_00000 TREB-1 88 ACGTCA 30 T00354 HBP-1 T00938 HBP-1b T01393 HBP- 88ACGTCA 1b(c1) T01394 HBP-1a(1) T01395 HBP-1a(c14) T02789 bZIP910 31T00051 ATF T00052 ATF-a T00053 ATF-adelta 86 YKRCGTCAT00054 ATF-like T00442 47-kDa CRE bind. prot. T00968 ATF-1 32T00111 c-Ets-1 T00112 c-Ets-1 T00114 c-Ets-1 54 97 SMGGAWGYT00115 c-Ets-1 68 T00684 PEA3 T00685 PEA3 T00686 PEA3 33T00506 MEF1 T00519 Myf-3 T00524 MyoD 69 GTCAGTTGT00525 MyoD T00526 MyoD T00527 MyoD T01128 MyoD

Surprisingly, the inventors noted the lack of any apparent difference inthe ability of Ad/sRep78 and Ad/dRep78 to grow in spite of theirdifferences in Rep expression level. It indicated that at least underthe control afforded by the tetracycline inducible system, the majorrole in inhibition of Ad replication was played by a sequence specificsignal and modification of that signal alone was sufficient tocompletely lift inhibition.

The ability of Ad/dRep78 and Ad/sRep78 to produce functional Rep78 wasconfirmed by an excision assay which depends on Rep's ability to cleaveat a folded AAV ITR (FIG. 2A). The inventors used a first generationAd/AAV carrying a single AAV ITR downstream of the transgene as asubstrate for cleavage. Cleavage at the ITR by Rep would result in therelease of an ˜8 Kb excision product. HEK 293 cells were co-infectedwith the substrate virus Ad/AAV FVIII and either Ad/sRep78 or Ad/dRep78in the presence or absence of doxycycline. Hirt DNA was prepared 48hours post infection and cleavage products were analyzed by southernblot with a substrate specific probe. Monomeric and dimeric excisionproducts that were dependent on the presence of Ad/sRep78 or Ad/dRep78were detected (FIGS. 2B and 2C). Leaky expression resulted in someexcision even in the absence of doxycycline, and a several fold increasein intensity seen with the addition of dox. Excision in C12 cells, aHeLa cell line derivative that inducibly expresses Rep and Cap (16) wasused as a positive control.

Example 4 Rep Protein Expression is not Required for Inhibition of AdReplication:

The fact that Ad/sRep78 and Ad/dRep78 replicate equally efficiently inspite of Ad/sRep78 expressing double the amount of Rep protein indicatesthat accumulation of Rep protein likely played no part in the inabilityof Ad/wtRep78 to replicate. However, the tetracycline inducible systemhas been shown to tightly regulate Rep protein expression in transfectedcells and as mentioned earlier, has been previously used for thesuccessful production of a helper dependent Ad carrying Rep78 (9).Further, the dependence of AAV on relative time of infection andrelative copy number to inhibit Ad replication has lead to a proposalthat the accumulation of Rep expression at the initial stages ofinfection is responsible for inhibition of Ad replication (11).Therefore, to truly understand the role of Rep78 protein expression inthe inhibition of Adenoviral replication, the inventors expressed themodified Rep ORFs under a constitutive 243 bp human U1-1 small nuclearRNA promoter (pH-U-1) (17). ΔE1ΔE3 Adenoviruses carrying the hu1-sRep78and hu1-dRep78 constructs were capable of normal rates of replication,with CPE observed 15 days after transfection. Viral yield (Pfu/cell)from Ad/hu1-sRep78 and Ad/hu1-dRep78 was comparable to both Ad/AAVFVIII, Ad/sRep78 and Ad/dRep78 (Table 1). These results prove that ahigh level of Rep78 protein expression can be tolerated by replicatingAdenoviruses and the dramatic inhibitory effects seen are mainly due tosignals within the sequence of the Rep ORF.

Example 5 Localization of Signal:

To localize the sequence specific inhibitory signal, the inventorsmodified the Scrambled Rep ORF to create—S (wt1) Rep. S (wt2) Rep, and S(wt3) Rep (FIG. 3A). In each of these constructs, using unique internalrestriction sites, sections representing about ⅓^(rd) (˜600 bps) of theentire Scrambled Rep sequence were replaced with that of WT Rep, inframe. These modified constructs were then inserted downstream of thetetracycline inducible promoter in place of scrambled Rep withinAd/sRep78, generating, Ad/S (wt1) Rep, Ad/S (wt2) Rep and Ad/S (wt3)Rep. The scrambled sequence was chosen to be modified as it expressessimilar amounts of Rep78 protein as the wild-type sequence. Presence ofthe entire inhibitory sequence within any of these ˜600 bp sectionsshould inhibit the replication of the virus carrying it. When linearizedand transfected into 293 cells, Ad/S (wt1) Rep and Ad/S (wt2) Rep bothshowed signs of viral replication shortly after primary infection andcomplete CPE within 15 days of transfection, and yielded titerscomparable to Ad/sRep78 (Table 1). Ad/S (wt3) Rep showed no signs ofreplication for up to 40 days post transfection, indicating that thesequence specific signal is localized within the 3′ 564 bps, in theregion encompassing bp 1623 to bp 2186 of the AAV2 genome.

To confirm that the inhibitory signal lay completely within the 3′ 564bp of the WT Rep78 sequence, the inventors then modified the WT Rep78sequence replacing the 3′ 564 bps alone with the corresponding ScrambledRep78 sequence, creating S (wt1,2) Rep (FIG. 3A). When inserteddownstream of the tetracycline inducible system within a firstgeneration Adenovirus, substitution of these 564 bps of the WT Repsequence with Scrambled Rep sequence alone was sufficient to liftinhibition and allow replication of the Adenovirus carrying it,comparable to Ad/sRep78 (Table 1).

In an alternative approach, the inventors used an algorithm to furthernarrow down the sequence specific inhibitory signal. The algorithmgenerates 4 different full length Rep encoding sequences (Design I, II,III and IV). The DNA sequence of each of these constructs is sub-dividedinto 14 segments, 123-135 bps in length, consisting of correspondingsequences from either the wild-type (WT) or scrambled (Scr) sequenceresulting in a ‘checkered’ pattern of segments (FIG. 3B). Thearrangement of segments in the 4 full length sequences is such that,when the 4 sequences are lined up, every column (consisting of thecorresponding segment in each of the 4 constructs) is unique. Whencloned into an expression cassette in a viral backbone, the presence ofthe sequence specific inhibitory signal in a particular segment willresult in all viruses with the WT sequence in that segment dying. Thus aunique pattern of viruses that live and die is associated with everypossible location of the sequence specific signal.

The 4 modified sequences were synthesized de novo by Genscript, USA andcloned downstream of the tetracycline inducible promoter, in place ofthe wt Rep cassette. These cassettes were inserted into first generationAds, generating Ad/Rep I, Ad/Rep II, Ad/Rep III and Ad/Rep TV (FIG. 3B).Each of these infectious clones were linearized and transfected into 293cells. Ad/Rep II, Ad/Rep III and Ad/Rep IV replicated and yielded titerscomparable to Ad/sRep78 (Table 1). Ad/Rep I showed no signs of viralreplication up to 50 days post transfection. The 135 bp segmentimplicated by the pattern of viruses capable of replication (Ad/Rep II,Ad/Rep III, and Ad/Rep IV) and incapable of replication (Ad/Rep I),encompassed bp 1782 to bp 1916 of the AAV2 genome (FIG. 3B—segmentindicated by an asterisk). Based on the arrangement of segments withinthe 4 sequences, some or all of the Rep sequence(s) that inhibits Adgrowth lies within this 135 bp fragment. This sequence lies well withinthe boundaries of bp 1623 to bp 2186 of the AAV2 genome identified bythe inability of S (wt3) Rep to grow.

Example 6 Additional Delineation of Rep Inhibitory Sequences.

Chimeric Rep genes were assembled by polynucleotide segment swapsencompassing discrete segments of wild-type or scrambled sequences, andviability established by adenoviral replication. The results are shownon FIGS. 14A-B-16A-D. These results demonstrate that scrambled sequenceswithin segment 11, segment 13 and segment 14 are capable of rescuing theinhibitory effect of the WT Rep ORF on Ad replication

REFERENCES

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All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described compositions and methods of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiment, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art and in fields related thereto are intended tobe within the scope of the following claims.

We claim:
 1. A method for reducing one or more symptoms of disease in amammalian subject, comprising administering a therapeutically effectiveamount of the vector of claim 46 to a mammalian subject in need oftherapy.
 2. The method of claim 1, further comprising detecting thepresence of at least a portion of the vector in a cell of the treatedsubject.
 3. The method of claim 1, wherein the recombinant nucleotidesequence further comprises a heterologous polynucleotide sequenceoperably linked to a first adeno-associated virus inverted terminalrepeat (AAV ITR).
 4. The method of claim 3, wherein the heterologouspolynucleotide sequence comprises a therapeutic sequences.
 5. The methodof claim 3, wherein the therapeutic sequences encodes one or both of adisease associated polypeptide and an antigen polypeptide.
 6. The methodof claim 5, wherein the therapeutic sequence encodes an antigenpolypeptide, and the method further comprises detecting an immuneresponse by the subject to the antigen polypeptide.